The many roads to mitochondrial dysfunction in neuroimmune and neuropsychiatric disorders

BackgroundMitochondrial dysfunction and defects in oxidative metabolism are a characteristic feature of many chronic illnesses not currently classified as mitochondrial diseases. Examples of such illnesses include bipolar disorder, multiple sclerosis, Parkinson’s disease, schizophrenia, depression, autism, and chronic fatigue syndrome.DiscussionWhile the majority of patients with multiple sclerosis appear to have widespread mitochondrial dysfunction and impaired ATP production, the findings in patients diagnosed with Parkinson’s disease, autism, depression, bipolar disorder schizophrenia and chronic fatigue syndrome are less consistent, likely reflecting the fact that these diagnoses do not represent a disease with a unitary pathogenesis and pathophysiology. However, investigations have revealed the presence of chronic oxidative stress to be an almost invariant finding in study cohorts of patients afforded each diagnosis. This state is characterized by elevated reactive oxygen and nitrogen species and/or reduced levels of glutathione, and goes hand in hand with chronic systemic inflammation with elevated levels of pro-inflammatory cytokines.SummaryThis paper details mechanisms by which elevated levels of reactive oxygen and nitrogen species together with elevated pro-inflammatory cytokines could conspire to pave a major road to the development of mitochondrial dysfunction and impaired oxidative metabolism seen in many patients diagnosed with these disorders.

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title: Mitochondrial dysfunctions in Myalgic Encephalomyelitis / chronic fatigue syndrome explained by activated immuno-inflammatory, oxidative and nitrosative stress pathways
abstract: Myalgic encephalomyelitis / chronic fatigue syndrome (ME/cfs) is classified by the World Health Organization as a disorder of the central nervous system. ME/cfs is an neuro-immune disorder accompanied by chronic low-grade inflammation, increased levels of oxidative and nitrosative stress (O&NS), O&NS-mediated damage to fatty acids, DNA and proteins, autoimmune reactions directed against neoantigens and brain disorders. Mitochondrial dysfunctions have been found in ME/cfs, e.g. lowered ATP production, impaired oxidative phosphorylation and mitochondrial damage. This paper reviews the pathways that may explain mitochondrial dysfunctions in ME/cfs. Increased levels of pro-inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-α, and elastase, and increased O&NS may inhibit mitochondrial respiration, decrease the activities of the electron transport chain and mitochondrial membrane potential, increase mitochondrial membrane permeability, interfere with ATP production and cause mitochondrial shutdown. The activated O&NS pathways may additionally lead to damage of mitochondrial DNA and membranes thus decreasing membrane fluidity. Lowered levels of antioxidants, zinc and coenzyme Q10, and ω3 polyunsaturated fatty acids in ME/cfs may further aggravate the activated immuno-inflammatory and O&NS pathways. Therefore, it may be concluded that immuno-inflammatory and O&NS pathways may play a role in the mitochondrial dysfunctions and consequently the bioenergetic abnormalities seen in patients with ME/cfs. Defects in ATP production and the electron transport complex, in turn, are associated with an elevated production of superoxide and hydrogen peroxide in mitochondria creating adaptive and synergistic damage. It is argued that mitochondrial dysfunctions, e.g. lowered ATP production, may play a role in the onset of ME/cfs symptoms, e.g. fatigue and post exertional malaise, and may explain in part the central metabolic abnormalities observed in ME/cfs, e.g. glucose hypometabolism and cerebral hypoperfusion.

title: Methylenetetrahydrofolate reductase polymorphisms C677T and risk of autism in the Chinese Han population.
abstract: Causes of autism are still unknown. Some studies have shown that autism might be associated with metabolic abnormalities in the folate/homocysteine pathway, which is involved in DNA methylation, thus altering gene expression. The association between the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphisms and the risk of autism is still controversial and ambiguous. The purpose of this study was to examine the effect of the MTHFR C677T polymorphism on the autism risk in the Chinese Han population. A population-based case-control study was conducted in 186 children with autism and 186 controls. The MTHFR C677T polymorphisms were determined by using a polymerase chain reaction-restriction fragment length polymorphism assay. The frequency of genotype MTHFR 677TT in children with autism (16.1%) was significantly higher (odds ratio [OR]=2.04; 95% confidence interval [CI]=1.07, 3.89; p=0.03] than those in controls (8.6%). When stratifying by select-item scores on the Autism Diagnostic Interview-Revised, it was found that children with current overactivity had a significantly higher frequency of the MTHFR 677TT genotype (OR=2.77, 95% CI=1.17, 6.60; p=0.02) than those without. This study suggested that MTHFR C677T is a risk factor of autism in Chinese Han children.

title: Sjøgren's syndrome-associated oxidative stress and mitochondrial dysfunction: Prospects for chemoprevention trials
abstract: An involvement of oxidative stress (OS) was found in recent studies of Sjøgren's syndrome (SS) that reported significant changes in protein oxidation, myeloperoxidase activity, TNF-α, nitrotyrosine, and GSH levels in plasma from SS patients. Excess levels of OS markers, as oxidative DNA damage and propanoyl-lysine, were reported in saliva from SS patients. Previous reports concurred with a role of OS in SS pathogenesis, by showing a decreased expression of antioxidant activities in conjunctival epithelial cells of SS patients and in parotid gland tissue samples from SS patients. A link between OS and mitochondrial dysfunction (MDF) is recognized both on the grounds of the established role of mitochondria in reactive oxygen species (ROS) formation and by the occurrence of MDF in a set of OS-related disorders. Earlier studies detected mitochondrial alterations in cells from SS patients, related to the action of antimitochondrial autoantibodies, and affecting specific mitochondrial activities. Thus, a link between MDF and OS may be postulated in SS, prompting studies aimed at elucidating SS pathogenesis and in the prospect of chemoprevention trials in SS clinical management.

title: Oxidative stress modulates mitochondrial failure and cyclophilin D function in X-linked adrenoleukodystrophy
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title: Assessment of mitochondrial dysfunction in lymphocytes of patients with systemic lupus erythematosus.
abstract: Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within the immune system. Activation, proliferation, or death of cells of the immune system is dependent on controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (∆ψ (m)) reflects the energy stored in the electrochemical gradient across the inner mitochondrial membrane which, in turn, is used by F(0)F(1)-ATPase to convert ADP to ATP during oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion represent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients with SLE exhibit elevated ∆ψ (m), i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alkalinization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP. Increased production of nitric oxide has been identified as a cause of MHP and increased mitochondrial biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox--sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key sensor of MHP and mediator of enhanced Ca(2+) flux in lupus T cells.

title: Myalgic encephalomyelitis/chronic fatigue syndrome and encephalomyelitis disseminata/multiple sclerosis show remarkable levels of similarity in phenomenology and neuroimmune characteristics
abstract: Background‘Encephalomyelitis disseminata’ (multiple sclerosis) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are both classified as diseases of the central nervous system by the World Health Organization. This review aims to compare the phenomenological and neuroimmune characteristics of MS with those of ME/CFS.DiscussionThere are remarkable phenomenological and neuroimmune overlaps between both disorders. Patients with ME/CFS and MS both experience severe levels of disabling fatigue and a worsening of symptoms following exercise and resort to energy conservation strategies in an attempt to meet the energy demands of day-to-day living. Debilitating autonomic symptoms, diminished cardiac responses to exercise, orthostatic intolerance and postural hypotension are experienced by patients with both illnesses. Both disorders show a relapsing-remitting or progressive course, while infections and psychosocial stress play a large part in worsening of fatigue symptoms. Activated immunoinflammatory, oxidative and nitrosative (O+NS) pathways and autoimmunity occur in both illnesses. The consequences of O+NS damage to self-epitopes is evidenced by the almost bewildering and almost identical array of autoantibodies formed against damaged epitopes seen in both illnesses. Mitochondrial dysfunctions, including lowered levels of ATP, decreased phosphocreatine synthesis and impaired oxidative phosphorylation, are heavily involved in the pathophysiology of both MS and ME/CFS. The findings produced by neuroimaging techniques are quite similar in both illnesses and show decreased cerebral blood flow, atrophy, gray matter reduction, white matter hyperintensities, increased cerebral lactate and choline signaling and lowered acetyl-aspartate levels.SummaryThis review shows that there are neuroimmune similarities between MS and ME/CFS. This further substantiates the view that ME/CFS is a neuroimmune illness and that patients with MS are immunologically primed to develop symptoms of ME/CFS.

title: Parkinson’s Disease: A Complex Interplay of Mitochondrial DNA Alterations and Oxidative Stress
abstract: Parkinson’s disease (PD) is one of the most common age-related neurodegenerative diseases. This pathology causes a significant loss of dopaminergic neurons in the Substantia Nigra. Several reports have claimed a role of defective nuclear and mitochondrial DNA repair pathways in PD etiology, in particular, of the Base Excision Repair (BER) system. In addition, recent findings, related to PD progression, indicate that oxidative stress pathways involving c-Abl and GST could also be implicated in this pathology. This review focuses on recently described networks most likely involved in an integrated manner in the course of PD.

title: Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism
abstract: Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders that are defined solely on the basis of behavioral observations. Therefore, ASD has traditionally been framed as a behavioral disorder. However, evidence is accumulating that ASD is characterized by certain physiological abnormalities, including oxidative stress, mitochondrial dysfunction and immune dysregulation/inflammation. While these abnormalities have been reported in studies that have examined peripheral biomarkers such as blood and urine, more recent studies have also reported these abnormalities in brain tissue derived from individuals diagnosed with ASD as compared to brain tissue derived from control individuals. A majority of these brain tissue studies have been published since 2010. The brain regions found to contain these physiological abnormalities in individuals with ASD are involved in speech and auditory processing, social behavior, memory, and sensory and motor coordination. This manuscript examines the evidence linking oxidative stress, mitochondrial dysfunction and immune dysregulation/inflammation in the brain of ASD individuals, suggesting that ASD has a clear biological basis with features of known medical disorders. This understanding may lead to new testing and treatment strategies in individuals with ASD.

title: Peripheral oxidative stress in relapsing-remitting multiple sclerosis.
abstract: To evaluate levels of oxidative stress in blood samples in patients with relapsing-remitting MS (RR-MS).



Peripheral blood samples were collected from 24 RR-MS patients and 15 healthy controls. Levels of the following were measured: carbonylated proteins, 8-hydroxy-2'deoxyguanosine (8OHdG), total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd), glutathione-S-transferase (GST), myeloperoxidase (MPO), antioxidant gap, total antioxidant capacity (PAO), global oxidative stress (GOS), serum vascular cell adhesion molecule-1 (sVCAM-1) and serum inter-cellular adhesion molecule 1 (sICAM-1).



Values for carbonylated proteins, 8OHdG, total glutathione, GSH, GSH/GSSG ratio, SOD, GRd and GOS were significantly higher in RR-MS patients than in healthy controls. By contrast, PAO, GSSG, GPx and GST were lower in RR-MS patients.



Oxidative stress plays a major role in MS, and is observed prior to relapse.

title: A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis
abstract: In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination—a hallmark of multiple sclerosis—is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.

title: Oxidative stress in multiple sclerosis patients in clinical remission: Association with the expanded disability status scale
abstract: Increased levels of oxidative stress markers and/or decreased levels of antioxidant molecules have been described in patients with multiple sclerosis (MS). This imbalance has been implicated in demyelination and axonal damage. The aims of this study were to evaluate oxidative stress in MS patients and to verify its correlation with disability as assessed by the expanded disability status scale (EDSS). This case-controlled study included 91 patients with relapsing-remitting multiple sclerosis (RR-MS) and 196 healthy individuals matched by age, gender, ethnicity, smoking status, and body mass index. Oxidative stress was evaluated by tert-butyl hydroperoxide-initiated chemiluminescence (CL-LOOH), carbonyl protein, nitric oxide metabolites (NOx), total radical-trapping antioxidant parameter (TRAP), sulfhydryl groups of proteins and serum uric acid levels. MS patients exhibited higher plasma levels of CL-LOOH (p<0.0001) and carbonyl protein (p=0.0081), and lower plasma levels of NOx (p<0.0001), TRAP (p=0.0088), and sulfhydryl groups (p=0.0003) compared to the control subjects. A multivariate analysis showed an association between oxidative markers and the presence of MS. Patients with an EDSS >3.5 showed higher CL-LOOH than control subjects (p=0.0093). A positive correlation was observed between CL-LOOH and EDSS (r=0.3244, p=0.0026) and between carbonyl protein and EDSS (r=0.3012, p=0.0041). These results demonstrate that oxidative stress plays an important role in the physiopathology of MS progression.

title: The involvement of mitochondria in the pathogenesis of multiple sclerosis
abstract: Multiple sclerosis is an immune-mediated disorder of the central nervous system. Major pathological characteristics include the loss of oligodendrocytes, demyelination and neuroaxonal depletion in association with inflammation. The complex pathophysiology of tissue loss is only partially understood. Here we discuss a variety of mitochondrion-driven mechanisms involved in immune regulation, oligodendrocyte depletion and neurodegeneration. The recognition of a mitochondrial link between inflammation and neurodegeneration underscores the importance of an early aggressive intervention for halting inflammation and preventing neurodegeneration, and identifies the mitochondrion as a potential target in neuroprotection.

title: Lipopolysaccharide-Induced Weakness in the Preterm Diaphragm Is Associated with Mitochondrial Electron Transport Chain Dysfunction and Oxidative Stress
abstract: Diaphragmatic contractility is reduced in preterm lambs after lipopolysaccharide (LPS) exposure in utero. The mechanism of impaired fetal diaphragm contractility after LPS exposure is unknown. We hypothesise that in utero exposure to LPS induces a deficiency of mitochondrial complex activity and oxidative damage in the fetal diaphragm. To test this hypothesis, we used a well-established preterm ovine model of chorioamnionitis: Pregnant ewes received intra-amniotic (IA) saline or 10 mg LPS, at 2 d or 7 d prior to surgical delivery at 121 d GA (term = 150 d). The fetus was killed humanely immediately after delivery for tissue sampling. Mitochondrial fractions were prepared from the isolated diaphragm and mitochondrial electron transfer chain activities were evaluated using enzymatic assays. Oxidative stress was investigated by quantifying mitochondrial oxidative protein levels and determining antioxidant gene and protein (catalase, superoxide dismutase 2 and glutathione peroxidase 1) expression. The activity of the erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signalling pathway was examined by quantifying the Nrf2 protein content of cell lysate and nuclear extract. A 2 d LPS exposure in utero significantly decreased electron transfer chain complex II and IV activity (p<0.05). A 7 d LPS exposure inhibited superoxide dismutase 2 and catalase expression at gene and protein levels, and Nrf2 pathway activity (p<0.05) compared with control and 2 d LPS groups, respectively. Diaphragm mitochondria accumulated oxidised protein after a 7 d LPS exposure. We conclude that intrauterine exposure to LPS induces mitochondrial oxidative stress and electron chain dysfunction in the fetal diaphragm, that is further exacerbated by impairment of the antioxidant signalling pathway and decreased antioxidant activity.

title: Oxidative Stress Induces Mitochondrial Dysfunction in a Subset of Autism Lymphoblastoid Cell Lines in a Well-Matched Case Control Cohort
abstract: There is increasing recognition that mitochondrial dysfunction is associated with the autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction or how mitochondrial abnormalities might interact with other physiological disturbances associated with autism, such as oxidative stress. In the current study we used respirometry to examine reserve capacity, a measure of the mitochondrial ability to respond to physiological stress, in lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) as well as age and gender-matched control LCLs. We demonstrate, for the first time, that LCLs derived from children with AD have an abnormal mitochondrial reserve capacity before and after exposure to increasingly higher concentrations of 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases intracellular reactive oxygen species (ROS). Specifically, the AD LCLs exhibit a higher reserve capacity at baseline and a sharper depletion of reserve capacity when ROS exposure is increased, as compared to control LCLs. Detailed investigation indicated that reserve capacity abnormalities seen in AD LCLs were the result of higher ATP-linked respiration and maximal respiratory capacity at baseline combined with a marked increase in proton leak respiration as ROS was increased. We further demonstrate that these reserve capacity abnormalities are driven by a subgroup of eight (32%) of 25 AD LCLs. Additional investigation of this subgroup of AD LCLs with reserve capacity abnormalities revealed that it demonstrated a greater reliance on glycolysis and on uncoupling protein 2 to regulate oxidative stress at the inner mitochondria membrane. This study suggests that a significant subgroup of AD children may have alterations in mitochondrial function which could render them more vulnerable to a pro-oxidant microenvironment derived from intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxicants. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors.

title: Bench-to-bedside review: Targeting antioxidants to mitochondria in sepsis
abstract: Development of organ dysfunction associated with sepsis is now accepted to be due at least in part to oxidative damage to mitochondria. Under normal circumstances, complex interacting antioxidant defense systems control oxidative stress within mitochondria. However, no studies have yet provided conclusive evidence of the beneficial effect of antioxidant supplementation in patients with sepsis. This may be because the antioxidants are not accumulating in the mitochondria, where they are most needed. Antioxidants can be targeted selectively to mitochondria by several means. This review describes the in vitro studies and animal models of several diseases involving oxidative stress, including sepsis, in which antioxidants targeted at mitochondria have shown promise, and the future implications for such approaches in patients.

title: Oxidative stress and mitochondrial dysfunction in sepsis.
abstract: Sepsis-related organ dysfunction remains the most common cause of death in the intensive care unit (ICU), despite advances in healthcare and science. Marked oxidative stress as a result of the inflammatory responses inherent with sepsis initiates changes in mitochondrial function which may result in organ damage. Normally, a complex system of interacting antioxidant defences is able to combat oxidative stress and prevents damage to mitochondria. Despite the accepted role that oxidative stress-mediated injury plays in the development of organ failure, there is still little conclusive evidence of any beneficial effect of systemic antioxidant supplementation in patients with sepsis and organ dysfunction. It has been suggested, however, that antioxidant therapy delivered specifically to mitochondria may be useful.

title: The Glutathione System: A New Drug Target in Neuroimmune Disorders
abstract: Glutathione (GSH) has a crucial role in cellular signaling and antioxidant defenses either by reacting directly with reactive oxygen or nitrogen species or by acting as an essential cofactor for GSH S-transferases and glutathione peroxidases. GSH acting in concert with its dependent enzymes, known as the glutathione system, is responsible for the detoxification of reactive oxygen and nitrogen species (ROS/RNS) and electrophiles produced by xenobiotics. Adequate levels of GSH are essential for the optimal functioning of the immune system in general and T cell activation and differentiation in particular. GSH is a ubiquitous regulator of the cell cycle per se. GSH also has crucial functions in the brain as an antioxidant, neuromodulator, neurotransmitter, and enabler of neuron survival. Depletion of GSH leads to exacerbation of damage by oxidative and nitrosative stress; hypernitrosylation; increased levels of proinflammatory mediators and inflammatory potential; dysfunctions of intracellular signaling networks, e.g., p53, nuclear factor-κB, and Janus kinases; decreased cell proliferation and DNA synthesis; inactivation of complex I of the electron transport chain; activation of cytochrome c and the apoptotic machinery; blockade of the methionine cycle; and compromised epigenetic regulation of gene expression. As such, GSH depletion has marked consequences for the homeostatic control of the immune system, oxidative and nitrosative stress (O&NS) pathways, regulation of energy production, and mitochondrial survival as well. GSH depletion and concomitant increase in O&NS and mitochondrial dysfunctions play a role in the pathophysiology of diverse neuroimmune disorders, including depression, myalgic encephalomyelitis/chronic fatigue syndrome and Parkinson’s disease, suggesting that depleted GSH is an integral part of these diseases. Therapeutical interventions that aim to increase GSH concentrations in vivo include N-acetyl cysteine; Nrf-2 activation via hyperbaric oxygen therapy; dimethyl fumarate; phytochemicals, including curcumin, resveratrol, and cinnamon; and folate supplementation.

title: NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury
abstract: Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and infiltrated macrophages, suggesting the assembly of functional complexes. Our data suggest that the inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in demyelination and free radical-mediated tissue injury in the pathogenesis of multiple sclerosis.

title: The role of oxidative stress in the
pathogenesis of multiple sclerosis: The need for effective
antioxidant therapy
abstract: Accumulating data indicate that oxidative stress (OS)
plays a major role in the pathogenesis of multiple sclerosis
(MS). Reactive oxygen species (ROS), leading to OS, generated in
excess primarily by macrophages, have been implicated as
mediators of demyelination and axonal damage in both MS and
experimental autoimmune encephalomyelitis (EAE), its animal
model. ROS cause damage to cardinal cellular components such as
lipids, proteins and nucleic acids (e. g., RNA, DNA), resulting
in cell death by necrosis or apoptosis. In addition, weakened
cellular antioxidant defense systems in the central nervous
system (CNS) in MS, and its vulnerability to ROS effects may
increase damage. Thus, treatment with antioxidants might
theoretically prevent propagation of tissue damage and improve
both survival and neurological outcome. Indeed, several
experimental studies have been performed to see whether dietary
intake of several antioxidants prevents or reduces the
progression of EAE. Although a few antioxidants showed some
efficacy in these studies, little information is available on
the effect of treatments with such compounds in patients with
MS. Well-designed clinical studies using antioxidant intake, as
well as investigations based on larger cohorts studied over a
longer periods of time, are needed in order to assess whether
antioxidant intake together with other conventional treatments,
might be beneficial in treating MS.

title: Mitochondrial generation of free radicals and hypoxic signaling
abstract: Most reactive oxygen species (ROS) are generated in cells by the mitochondrial respiratory chain. Mitochondrial ROS production is modulated largely by the rate of electron flow through respiratory chain complexes. Recently, it has become clear that under hypoxic conditions, the mitochondrial respiratory chain also produces nitric oxide (NO), which can generate other reactive nitrogen species (RNS). Although excess ROS and RNS can lead to oxidative and nitrosative stress, moderate to low levels of both function in cellular signaling pathways. Especially important are the roles of these mitochondrially generated free radicals in hypoxic signaling pathways, which have important implications for cancer, inflammation and a variety of other diseases.

title: Targeting microglia-mediated neurotoxicity: the potential of NOX2 inhibitors
abstract: Microglia are key sentinels of central nervous system health, and their dysfunction has been widely implicated in the progressive nature of neurodegenerative diseases. While microglia can produce a host of factors that are toxic to neighboring neurons, NOX2 has been implicated as a common and essential mechanism of microglia-mediated neurotoxicity. Accumulating evidence indicates that activation of the NOX2 enzyme complex in microglia is neurotoxic, both through the production of extracellular reactive oxygen species that damage neighboring neurons as well as the initiation of redox signaling in microglia that amplifies the pro-inflammatory response. More specifically, evidence supports that NOX2 redox signaling enhances microglial sensitivity to pro-inflammatory stimuli, and amplifies the production of neurotoxic cytokines, to promote chronic and neurotoxic microglial activation. Here, we describe the evidence denoting the role of NOX2 in microglia-mediated neurotoxicity with an emphasis on Alzheimer’s and Parkinson’s disease, describe available inhibitors that have been tested, and detail evidence of the neuroprotective and therapeutic potential of targeting this enzyme complex to regulate microglia.

title: Role of Nox family NADPH oxidases in host defense.
abstract: The phagocytic NADPH oxidase is recognized as a critical component of innate immunity, responsible for generation of microbicidal reactive oxygen species (ROS). This enzyme is one representative of the Nox family of oxidases (Nox1-Nox5, Duox1, and Duox2) that exhibit diverse expression patterns and appear to serve a variety of functions related to ROS generation. Mounting evidence now suggests that several of these novel oxidases also serve in host defense, particularly those showing high expression along epithelial surfaces exposed to the external environment. Within these sites, Nox enzymes tend to be located on apical cell surfaces and release ROS into extracellular environments, where they can be used by known antimicrobial peroxidases. Moreover, microbial factors were shown in several cases to cause higher ROS production, either by direct oxidase activation or by inducing higher oxidase expression. Several oxidases are also induced by immune cytokines, including interferon-gamma, interleukin (IL)-4, and IL-13. Although most of the evidence supporting host defense roles for mammalian nonphagocytic oxidases remains circumstantial, recent evidence indicates that Drosophila Duox plays a role in host resistance to infection. Finally, oxidative defense against invading pathogens appears to be an ancient protective mechanism, because related oxidases are known to participate in disease resistance in plants.

title: Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis
abstract: Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS.  XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies.  We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination.  These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.

title: Peroxynitrite, a potent macrophage‐derived oxidizing cytotoxin to combat invading pathogens
abstract: Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill micro‐organisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage‐derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens.

title: Oxidative stress, inflammation, and cancer: how are they linked?
abstract: Extensive research during the past 2 decades has revealed the mechanism by which continued oxidative stress can lead to chronic inflammation, which in turn could mediate most chronic diseases including cancer, diabetes, and cardiovascular, neurological, and pulmonary diseases. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules. How oxidative stress activates inflammatory pathways leading to transformation of a normal cell to tumor cell, tumor cell survival, proliferation, chemoresistance, radioresistance, invasion, angiogenesis, and stem cell survival is the focus of this review. Overall, observations to date suggest that oxidative stress, chronic inflammation, and cancer are closely linked.

title: Causal link between oxidative stress, inflammation, and hypertension.
abstract: Hypertension (HTN) is a major cause of stroke, left ventricular hypertrophy, congestive heart failure, arteriosclerosis, end-stage renal disease, and peripheral vascular disease. Oxidative stress and its constant companion, inflammation, play a critical part in the pathogenesis of many acute and chronic illnesses including HTN and its long-term complications. There is compelling evidence that oxidative stress, inflammation, and HTN are involved in a self-perpetuating vicious cycle which, if not interrupted, culminates in progressive target organ injury and dysfunction. This article is intended to review the available evidence for the role of oxidative stress and inflammation in the pathogenesis of HTN. In addition, evidence will be presented to demonstrate the role of HTN in the pathogenesis of oxidative stress and inflammation. Finally, evidence for participation of tissue angiotensin system in the vicious cycle of oxidative stress, inflammation, and HTN will be presented, and the approach to treatment of HTN-associated oxidative stress will be discussed.

title: Intraphagosomal Peroxynitrite as a Macrophage-derived Cytotoxin against Internalized Trypanosoma cruzi
abstract: Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO− + ONOOH) formation, a strong oxidant arising from the reaction of •NO with superoxide radical (O2̇̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O2̇̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained •NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when •NO and O2̇̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.

title: Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer.
abstract: Chronic inflammation is a pathological condition characterized by continued active inflammation response and tissue destruction. Many of the immune cells including macrophages, neutrophils and eosinophils are involved directly or by production of inflammatory cytokine production in pathology of chronic inflammation. From literatures, it is appear that there is a general concept that chronic inflammation can be a major cause of cancers and express aging processes. Moreover, many studies suggest that chronic inflammation could have serious role in wide variety of age-related diseases including diabetes, cardiovascular and autoimmune diseases. Inflammatory process induces oxidative stress and reduces cellular antioxidant capacity. Overproduced free radicals react with cell membrane fatty acids and proteins impairing their function permanently. In addition, free radicals can lead to mutation and DNA damage that can be a predisposing factor for cancer and age-related disorders. This article reviews the antioxidant defense systems, free radicals production and their role in cancer and age related diseases and also some of the recent patent relevant to the field. Study of the role of free radicals in human diseases can help the investigators to consider the antioxidants as proper agents in preventive medicine, especially for cancer and aging processes.

title: Immunology and Oxidative Stress in Multiple Sclerosis: Clinical and Basic Approach
abstract: Multiple sclerosis (MS) exhibits many of the hallmarks of an inflammatory autoimmune disorder including breakdown of the blood-brain barrier (BBB), the recruitment of lymphocytes, microglia, and macrophages to lesion sites, the presence of multiple lesions, generally being more pronounced in the brain stem and spinal cord, the predominantly perivascular location of lesions, the temporal maturation of lesions from inflammation through demyelination, to gliosis and partial remyelination, and the presence of immunoglobulin in the central nervous system and cerebrospinal fluid. Lymphocytes activated in the periphery infiltrate the central nervous system to trigger a local immune response that ultimately damages myelin and axons. Pro-inflammatory cytokines amplify the inflammatory cascade by compromising the BBB, recruiting immune cells from the periphery, and activating resident microglia. inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in the demyelination and free radical-mediated tissue injury in the pathogenesis of MS. The inflammatory environment in demyelinating lesions leads to the generation of oxygen- and nitrogen-free radicals as well as proinflammatory cytokines which contribute to the development and progression of the disease. Inflammation can lead to oxidative stress and vice versa. Thus, oxidative stress and inflammation are involved in a self-perpetuating cycle.

title: Role of the Toll Like Receptor (TLR) Radical Cycle in Chronic Inflammation: Possible Treatments Targeting the TLR4 Pathway
abstract: Activation of the Toll-like receptor 4 (TLR4) complex, a receptor of the innate immune system, may underpin the pathophysiology of many human diseases, including asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation. TLRs are pattern recognition receptors that recognize damage-associated molecular patterns and pathogen-associated molecular patterns, including lipopolysaccharide (LPS) from gram-negative bacteria. Here we focus on the environmental factors, which are known to trigger TLR4, e.g., ozone, atmosphere particulate matter, long-lived reactive oxygen intermediate, pentachlorophenol, ionizing radiation, and toluene. Activation of the TLR4 pathways may cause chronic inflammation and increased production of reactive oxygen and nitrogen species (ROS/RNS) and oxidative and nitrosative stress and therefore TLR-related diseases. This implies that drugs or substances that modify these pathways may prevent or improve the abovementioned diseases. Here we review some of the most promising drugs and agents that have the potential to attenuate TLR-mediated inflammation, e.g., anti-LPS strategies that aim to neutralize LPS (synthetic anti-LPS peptides and recombinant factor C) and TLR4/MyD88 antagonists, including eritoran, CyP, EM-163, epigallocatechin-3-gallate, 6-shogaol, cinnamon extract, N-acetylcysteine, melatonin, and molecular hydrogen. The authors posit that activation of the TLR radical (ROS/RNS) cycle is a common pathway underpinning many “civilization” disorders and that targeting the TLR radical cycle may be an effective method to treat many inflammatory disorders.

title: Mechanisms of Oxidative Damage in Multiple Sclerosis and a Cell Therapy Approach to Treatment
abstract: Although significant advances have recently been made in the understanding and treatment of multiple sclerosis, reduction of long-term disability remains a key goal. Evidence suggests that inflammation and oxidative stress within the central nervous system are major causes of ongoing tissue damage in the disease. Invading inflammatory cells, as well as resident central nervous system cells, release a number of reactive oxygen and nitrogen species which cause demyelination and axonal destruction, the pathological hallmarks of multiple sclerosis. Reduction in oxidative damage is an important therapeutic strategy to slow or halt disease processes. Many drugs in clinical practice or currently in trial target this mechanism. Cell-based therapies offer an alternative source of antioxidant capability. Classically thought of as being important for myelin or cell replacement in multiple sclerosis, stem cells may, however, have a more important role as providers of supporting factors or direct attenuators of the disease. In this paper we focus on the antioxidant properties of mesenchymal stem cells and discuss their potential importance as a cell-based therapy for multiple sclerosis.

title: Microglial priming in neurodegenerative disease
abstract: Under physiological conditions, the number and function of microglia—the resident macrophages of the CNS—is tightly controlled by the local microenvironment. In response to neurodegeneration and the accumulation of abnormally folded proteins, however, microglia multiply and adopt an activated state—a process referred to as priming. Studies using preclinical animal models have shown that priming of microglia is driven by changes in their microenvironment and the release of molecules that drive their proliferation. Priming makes the microglia susceptible to a secondary inflammatory stimulus, which can then trigger an exaggerated inflammatory response. The secondary stimulus can arise within the CNS, but in elderly individuals, the secondary stimulus most commonly arises from a systemic disease with an inflammatory component. The concept of microglial priming, and the subsequent exaggerated response of these cells to secondary systemic inflammation, opens the way to treat neurodegenerative diseases by targeting systemic disease or interrupting the signalling pathways that mediate the CNS response to systemic inflammation. Both lifestyle changes and pharmacological therapies could, therefore, provide efficient means to slow down or halt neurodegeneration.

title: Microglia and neurodegeneration: The role of systemic inflammation
abstract: It is well accepted that CNS inflammation has a role in the progression of chronic neurodegenerative disease, although the mechanisms through which this occurs are still unclear. The inflammatory response during most chronic neurodegenerative disease is dominated by the microglia and mechanisms by which these cells contribute to neuronal damage and degeneration are the subject of intense study. More recently it has emerged that systemic inflammation has a significant role to play in the progression of these diseases. Well‐described adaptive pathways exist to transduce systemic inflammatory signals to the brain, but activation of these pathways appears to be deleterious to the brain if the acute insult is sufficiently robust, as in severe sepsis, or sufficiently prolonged, as in repeated stimulation with robust doses of inflammogens such as lipopolysaccharide (LPS). Significantly, moderate doses of inflammogens produce new pathology in the brain and exacerbate or accelerate features of disease when superimposed upon existing pathology or in the context of genetic predisposition. It is now apparent in multiple chronic disease states, and in ageing, that microglia are primed by prior pathology, or by genetic predisposition, to respond more vigorously to subsequent inflammatory stimulation, thus transforming an adaptive CNS inflammatory response to systemic inflammation, into one that has deleterious consequences for the individual. In this review, the preclinical and clinical evidence supporting a significant role for systemic inflammation in chronic neurodegenerative diseases will be discussed. Mechanisms by which microglia might effect neuronal damage and dysfunction, as a consequence of systemic stimulation, will be highlighted.

title: Role of peripheral immune response in microglia activation and regulation of brain chemokine and proinflammatory cytokine responses induced during VSV encephalitis
abstract: We report herein that neuroinvasion by vesicular stomatitis virus (VSV) activates microglia and induces a peripheral dendritic cell (DC)-dependent inflammatory response in the central nervous system (CNS). VSV neuroinvasion rapidly induces multiple brain chemokine and proinflammatory cytokine mRNAs that display bimodal kinetics. Peripheral DC ablation or T cell depletion suppresses the second wave of this response demonstrating that infiltrating T cells are primarily responsible for the bimodal characteristics of this response. The robust infiltrate associated with VSV encephalitis likely depends on sustained production of brain CCL19 and CCR7 expression on infiltrating inflammatory cells.

title: Oxidative and Nitrosative Stress and Immune-Inflammatory Pathways in Patients with Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS)
abstract: Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome (CFS) has been classified as a disease of the central nervous system by the WHO since 1969. Many patients carrying this diagnosis do demonstrate an almost bewildering array of biological abnormalities particularly the presence of oxidative and nitrosative stress (O&NS) and a chronically activated innate immune system. The proposal made herein is that once generated chronically activated O&NS and immune-inflammatory pathways conspire to generate a multitude of self-sustaining and self-amplifying pathological processes which are associated with the onset of ME/CFS. Sources of continuous activation of O&NS and immune-inflammatory pathways in ME/CFS are chronic, intermittent and opportunistic infections, bacterial translocation, autoimmune responses, mitochondrial dysfunctions, activation of the Toll-Like Receptor Radical Cycle, and decreased antioxidant levels. Consequences of chronically activated O&NS and immune-inflammatory pathways in ME/CFS are brain disorders, including neuroinflammation and brain hypometabolism / hypoperfusion, toxic effects of nitric oxide and peroxynitrite, lipid peroxidation and oxidative damage to DNA, secondary autoimmune responses directed against disrupted lipid membrane components and proteins, mitochondrial dysfunctions with a disruption of energy metabolism (e.g. compromised ATP production) and dysfunctional intracellular signaling pathways. The interplay between all of these factors leads to self-amplifying feed forward loops causing a chronic state of activated O&NS, immune-inflammatory and autoimmune pathways which may sustain the disease.

title: A neuro-immune model of Myalgic Encephalomyelitis/Chronic fatigue syndrome
abstract: This paper proposes a neuro-immune model for Myalgic Encephalomyelitis/Chronic fatigue syndrome (ME/CFS). A wide range of immunological and neurological abnormalities have been reported in people suffering from ME/CFS. They include abnormalities in proinflammatory cytokines, raised production of nuclear factor-κB, mitochondrial dysfunctions, autoimmune responses, autonomic disturbances and brain pathology. Raised levels of oxidative and nitrosative stress (O&NS), together with reduced levels of antioxidants are indicative of an immuno-inflammatory pathology. A number of different pathogens have been reported either as triggering or maintaining factors. Our model proposes that initial infection and immune activation caused by a number of possible pathogens leads to a state of chronic peripheral immune activation driven by activated O&NS pathways that lead to progressive damage of self epitopes even when the initial infection has been cleared. Subsequent activation of autoreactive T cells conspiring with O&NS pathways cause further damage and provoke chronic activation of immuno-inflammatory pathways. The subsequent upregulation of proinflammatory compounds may activate microglia via the vagus nerve. Elevated proinflammatory cytokines together with raised O&NS conspire to produce mitochondrial damage. The subsequent ATP deficit together with inflammation and O&NS are responsible for the landmark symptoms of ME/CFS, including post-exertional malaise. Raised levels of O&NS subsequently cause progressive elevation of autoimmune activity facilitated by molecular mimicry, bystander activation or epitope spreading. These processes provoke central nervous system (CNS) activation in an attempt to restore immune homeostatsis. This model proposes that the antagonistic activities of the CNS response to peripheral inflammation, O&NS and chronic immune activation are responsible for the remitting-relapsing nature of ME/CFS. Leads for future research are suggested based on this neuro-immune model.

title: Immune Responses in Parkinson's Disease: Interplay between Central and Peripheral Immune Systems
abstract: The etiology of Parkinson's disease (PD) is complex and most likely involves numerous environmental and heritable risk factors. Recent studies establish that central and peripheral inflammation occurs in the prodromal stage of the disease and sustains disease progression. Aging, heritable risk factors, or environmental exposures may contribute to the initiation of central or peripheral inflammation. One emerging hypothesis is that inflammation plays a critical role in PD neuropathology. Increasing evidence suggest that activation of the peripheral immune system exacerbates the discordant central inflammatory response and synergistically drives neurodegeneration. We provide an overview of current knowledge on the temporal profile of central and peripheral immune responses in PD and discuss the potential synergistic effects of the central and peripheral inflammation in disease development. The understanding of the nature of the chronic inflammation in disease progression and the possible risk factors that contribute to altered central and peripheral immune responses will offer mechanistic insights into PD etiology and pathology and benefit the development of effective tailored therapeutics for human PD.

title: Parkinson's Disease and Systemic Inflammation
abstract: Peripheral inflammation triggers exacerbation in the central brain's ongoing damage in several neurodegenerative diseases. Systemic inflammatory stimulus induce a general response known as sickness behaviour, indicating that a peripheral stimulus can induce the synthesis of cytokines in the brain. In Parkinson's disease (PD), inflammation was mainly associated with microglia activation that can underlie the neurodegeneration of neurons in the substantia nigra (SN). Peripheral inflammation can transform the “primed” microglia into an “active” state, which can trigger stronger responses dealing with neurodegenerative processes. Numerous evidences show that systemic inflammatory processes exacerbate ongoing neurodegeneration in PD patient and animal models. Anti-inflammatory treatment in PD patients exerts a neuroprotective effect. In the present paper, we analyse the effect of peripheral infections in the etiology and progression in PD patients and animal models, suggesting that these peripheral immune challenges can exacerbate the symptoms in the disease.

title: Systemic Inflammation Modulates Fc Receptor Expression on Microglia during Chronic Neurodegeneration
abstract: Chronic neurodegeneration is a major worldwide health problem, and it has been suggested that systemic inflammation can accelerate the onset and progression of clinical symptoms. A possible explanation is that systemic inflammation “switches” the phenotype of microglia from a relatively benign to a highly aggressive and tissue-damaging phenotype. The current study investigated the molecular mechanism underlying this microglia phenotype “switching.” We show in mice with chronic neurodegeneration (ME7 prion model) that there is increased expression of receptors that have a key role in macrophage activation and associated signaling pathways, including TREM-2, Siglec-F, CD200R, and FcγRs. Systemic inflammation induced by LPS further increased protein levels of the activating FcγRIII and FcγRIV, but not of other microglial receptors, including the inhibitory FcγRII. In addition to these changes in receptor expression, IgG levels in the brain parenchyma were increased during chronic neurodegeneration, and these IgG levels further increased after systemic inflammation. γ-Chain–deficient mice show modified proinflammatory cytokine expression in the brain after systemic inflammation. We conclude that systemic inflammation during chronic neurodegeneration increases the expression levels of activating FcγR on microglia and thereby lowers the signaling threshold for Ab-mediated cell activation. At the same time, IgG influx into the brain could provide a cross-linking ligand resulting in excessive microglia activation that is detrimental to neurons already under threat by misfolded protein.

title: Contribution of systemic inflammation to chronic neurodegeneration
abstract: Systemic infection or inflammation gives rise to signals that communicate with the brain and leads to changes in metabolism and behaviour collectively known as sickness behaviour. In healthy young individuals, these changes are normally transient with no long-term consequences. The microglia are involved in the immune to brain signalling pathways. In the aged or diseased brain, the microglia have a primed phenotype as a consequence of changes in their local microenvironment. Systemic inflammation impacts on these primed microglia and switches them from a relatively benign to an aggressive phenotype with the enhanced synthesis of pro-inflammatory mediators. Recent evidence suggests that systemic inflammation contributes to the exacerbation of acute symptoms of chronic neurodegenerative disease and may accelerate disease progression. The normal homeostatic role that microglia play in signalling about systemic infections and inflammation becomes maladaptive in the aged and diseased brain and this offers a route to therapeutic intervention. Prompt treatment of systemic inflammation or blockade of signalling pathways from the periphery to the brain may help to slow neurodegeneration and improve the quality of life for individuals suffering from chronic neurodegenerative disease.

title: Neuroinflammation, oxidative stress and the pathogenesis of Alzheimer's disease.
abstract: Alzheimer's disease (AD) is the most common neurodegenerative disorder that affects the elderly. The increase of life-expectancy is transforming AD into a major health-care problem. AD is characterized by a progressive impairment of memory and other cognitive skills leading to dementia. The major pathogenic factor associated to AD seems to be amyloid-beta peptide (Aβ) oligomers that tend to accumulate extracellularly as amyloid deposits and are associated with reactive microglia and astrocytes as well as with degeneration of neuronal processes. The involvement of microglia and astrocytes in the onset and progress of neurodegenerative process in AD is becoming increasingly recognized, albeit it is commonly accepted that neuroinflammation and oxidative stress can have both detrimental and beneficial influences on the neural tissue. However, little is known about the interplay of microglia, astrocytes and neurons in response to Aβ, especially in the early phases of AD. This review discusses current knowledge about the involvement of neuroinflammation in AD pathogenesis, focusing on phenotypic and functional responses of microglia, astrocytes and neurons in this process. The abnormal production by glia cells of pro-inflammatory cytokines, chemokines and the complement system, as well as reactive oxygen and nitrogen species, can disrupt nerve terminals activity causing dysfunction and loss of synapses, which correlates with memory decline; these are phenomena preceding the neuronal death associated with late stages of AD. Thus, therapeutic strategies directed at controlling the activation of microglia and astrocytes and the excessive production of pro-inflammatory and pro-oxidant factors may be valuable to control neurodegeneration in dementia.

title: Peripheral cytokines profile in Parkinson’s disease
abstract: Higher levels of proinflammatory cytokines are found in Parkinson's disease (PD) patient's brains and inflammation is thought to be a major contributor to the neurodegeneration. During the inflammatory process, microglial release of proinflammatory cytokines act on the endothelium of blood-brain barrier (BBB) cells to stimulate upregulation of adhesion molecules. Consequently, this upregulation leads to the recruitment of passing T cells and monocytes, which express the counter receptors, that then go on to release more cytokines [Whitton, P.S., 2007. Inflammation as a causative factor in the aetiology of Parkinson's disease, Br.  Kortekaas, R., Leenders, K.L., Van Oostrom, J.C., Vaalburg, W., Bart, J., Willemsen, A.T., Hendrikse,  2005. Blood-brain barrier dysfunction in parkinsonian midbrain in vivo, . In addition, a systemic inflammatory response results in the production of cytokines which circulate in the blood and communicate with neurons within the brain. Thus, a central inflammatory reaction interacts with peripheral blood mononuclear cells (PBMCs) modulating immune activity. The present study investigates levels of production and expression of cyto/chemokines by PBMCs in PD patients. Basal and LPS-induced levels of MCP-1, RANTES, MIP-1alpha, IL-8, IFNgamma, IL-1beta and TNFalpha were significantly higher in PD patients than in HC subjects (p<0.001), as determined by RT-PCR and Elisa methods. Cyto/chemokine levels were significantly correlated with UPDRS III and H/Y stage (p<0.001). The Pearson's correlation coefficient (R) was also used to assess the strength of the relationship between NF-kappaBp65 levels and all studied cyto/chemokines and between NF-kappaBp65, UPDRS III and H/Y score in PD patients. The overall results strengthen and extend the knowledge of the peripheral dysregulation in the cytokine network associated with PD.

title: Peripheral chemo-cytokine profiles in Alzheimer's and Parkinson's diseases.
abstract: The identification of peripheral biomarkers for neurodegenerative diseases is required to improve the accuracy of clinical diagnosis and monitor both disease progression and response to treatments. The data reviewed in this paper suggest that, in neurodegenerative disease, cytokines are links between peripheral immune system and nervous system dysfunction.

title: Differential effects of fingolimod on B-cell populations in multiple sclerosis
abstract: Fingolimod is an oral drug approved for multiple sclerosis (MS) with an ability to trap central memory T cells in secondary lymphoid tissues; however, its variable effectiveness in individual patients indicates the need to evaluate its effects on other lymphoid cells.  To clarify the effects of fingolimod on B-cell populations in patients with MS.  We analysed blood samples from 9 fingolimod-treated and 19 control patients with MS by flow cytometry, to determine the frequencies and activation states of naive B cells, memory B cells, and plasmablasts.  The frequencies of each B-cell population in peripheral blood mononuclear cells (PBMC) were greatly reduced 2 weeks after starting fingolimod treatment. Detailed analysis revealed a significant reduction in activated memory B cells (CD38int-high), particularly those expressing Ki-67, a marker of cell proliferation. Also, we noted an increased proportion of activated plasmablasts (CD138+) among whole plasmablasts, in the patients treated with fingolimod.  The marked reduction of Ki-67+ memory B cells may be directly linked with the effectiveness of fingolimod in treating MS. In contrast, the relative resistance of CD138+ plasmablasts to fingolimod may be of relevance for understanding the differential effectiveness of fingolimod in individual patients.

title: Cellular sources of dysregulated cytokines in relapsing-remitting multiple sclerosis
abstract: BackgroundNumerous cytokines are implicated in the immunopathogenesis of multiple sclerosis (MS), but studies are often limited to whole blood (WB) or peripheral blood mononuclear cells (PBMCs), thereby omitting important information about the cellular origin of the cytokines. Knowledge about the relation between blood and cerebrospinal fluid (CSF) cell expression of cytokines and the cellular source of CSF cytokines is even more scarce.MethodsWe studied gene expression of a broad panel of cytokines in WB from relapsing-remitting multiple sclerosis (RRMS) patients in remission and healthy controls (HCs). Subsequently we determined the gene expression of the dysregulated cytokines in isolated PBMC subsets (CD4+, CD8+T-cells, NK-cells, B-cells, monocytes and dendritic cells) from RRMS patients and HCs and in CSF-cells from RRMS patients in clinical relapse and non-inflammatory neurological controls (NIND).ResultsRRMS patients had increased expression of IFN-gamma (IFNG), interleukin (IL) 1-beta (IL1B), IL7, IL10, IL12A, IL15, IL23, IL27, lymphotoxin-alpha (LTA) and lymphotoxin-beta (LTB) in WB. In PBMC subsets the main sources of pro-inflammatory cytokines were T- and B-cells, whereas monocytes were the most prominent source of immunoregulatory cytokines. In CSF-cells, RRMS patients had increased expression of IFNG and CD19 and decreased expression of IL10 and CD14 compared to NINDs. CD19 expression correlated with expression of IFNG, IL7, IL12A, IL15 and LTA whereas CD14 expression correlated with IL10 expression.ConclusionsUsing a systematic approach, we show that expression of pro-inflammatory cytokines in peripheral blood primarily originates from T- and B-cells, with an important exception of IFNG which is most strongly expressed by NK-cells. In CSF-cell studies, B-cells appear to be enriched in RRMS and associated with expression of pro-inflammatory cytokines; contrarily, monocytes are relatively scarce in CSF from RRMS patients and are associated with IL10 expression. Thus, our findings suggest a pathogenetic role of B-cells and an immunoregulatory role of monocytes in RRMS.

title: Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: Do cytokines trigger off exacerbations?
abstract: ABSTRACT‐ We have carried out a longitudinal study of interferon (IFN) and tumor necrosis factor (TNF) using a whole‐blood mitogen stimulation assay in 20 multiple sclerosis (MS) patients and in a healthy control group. We set up individual profiles and the results were quite constant for each individual, both in healthy donors and in the patients in remission. Before exacerbations, however, we found an increase of IFN‐γ and TNF production preceding clinical symptoms by a maximum of 2 weeks. In benign cases, the increase disappeared rapidly, even before the appearance of symptoms, whereas we found sequelae whenever the increase persisted during weeks. In chronic progressive patients, we frequently found intervening increases. It may be that IFN‐γ and TNF trigger off exacerbations at a very early stage and that these cytokines may also play a role in maintaining disease in chronic progressive and invalidating forms.

title: Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis
abstract: Interleukin (IL) 1 beta, tumor necrosis factor alpha (TNF alpha), and IL-6 are cytokines which mediate cellular responses during immune activation and inflammation. In multiple sclerosis (MS) they might be responsible for T-cell activation (IL-1 beta), for demyelination (TNF alpha), and for immunoglobulin (Ig) synthesis (IL-6) within the central nervous system. We studied IL-1 beta, TNF alpha, and IL-6 levels in the cerebrospinal fluid (CSF) of 34 patients with MS, 43 patients with non-inflammatory neurological diseases (NIND), and 19 patients with inflammatory neurological diseases (IND). IL-6 was found in the CSF of 29% of MS, 7% of NIND, and 47% of IND patients. TNF alpha was detected in the CSF of 23% of MS, 7% of NIND, and 29% of IND. CSF IL-6 and TNF alpha levels were significantly higher in MS and IND than in NIND. IL-1 beta was rarely detected in the CSF of any group. At least one cytokine was detected in 52% of MS CSF, 11% of NIND CSF, and 64% of IND CSF. In MS patients, no relationship was observed between the incidence or the amount of intrathecal IgG synthesis or oligoclonal bands and the presence of any cytokine. We also evaluated cytokine levels in paired sera from 11 MS and 13 NIND patients. Low levels of IL-6 were detected in most sera from MS and NIND patients. TNF alpha was detected in only two MS sera, and IL-1 beta was undetectable in any sample. Our results indicate that increased CSF levels of the cytokines IL-6 and TNF alpha occur frequently in MS and IND, but there is no obvious relationship to intrathecal Ig synthesis.

title: Review: cytokines and the pathogenesis of multiple sclerosis
abstract: Perivascular accumulation of mononuclear cells (MNCs) in the central nervous system (CNS) and high levels of myelin autoantigen‐reactive T cells in blood and further enriched in cerebrospinal fluid (CSF) are characteristic for multiple sclerosis (MS) and suggest a role for immunoregulatory cytokines in MS pathogenesis. The difficulties inherent to measurements of cytokine concentrations in body fluids have been partly overcome by adopting techniques allowing cytokine determinations on cellular level. MS is associated with the parallel up‐regulation of proinflammatory [interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), lymphotoxin‐α, and interleukin (IL)‐12] and immune response‐down‐regulating [transforming growth factor‐β (TGF‐β), IL‐10] cytokines systemically. A preferential up‐modulation of TNF‐α and lymphotoxin‐α is observed in clinical exacerbations and of TGF‐β and IL‐10 in remissions. The B cell‐stimulating IL‐4 and IL‐6 are also up‐regulated in MS, as is the cytolysis‐promoting perforin. Cytokine production is elevated to an even higher degree in the CSF than systemically, underlining the autonomy of the immune responses in the CSF. All cytokine abnormalities are demonstrable already in very early MS, manifested by acute unilateral optic neuritis associated with more than two MS‐like lesions on brain magnetic resonance imaging and oligoclonal IgG bands in CSF. The cytokine abnormalities hitherto observed are not MS specific, because they can be found in other inflammatory CNS diseases, e.g., aseptic meningitis and even noninflammatory neurological diseases like stroke. The influence on cytokine profiles, e.g., suppressing proinflammatory cytokines and promoting TGF‐β and IL‐10, could be an important way to identify new and promising treatments of MS.

title: Analysis of proinflammatory and anti-inflammatory cytokine serum concentrations in patients with multiple sclerosis by using a multiplexed immunoassay.
abstract: We examined cytokines and other inflammatory markers in serum samples from 833 patients with multiple sclerosis and 117 healthy control subjects. A multiplexed immunoassay was used to assess the concentrations of 13 cytokines/inflammatory markers: interferon (IFN)-γ; interleukins (ILs)-1β, 2, 4, 5, 6, 8, 10, 12, and 13; tumor necrosis factor (TNF)-α; IL-2 receptor; and soluble CD40 ligand. Significant increases between patients and control subjects were found for IFN-γ (mean, 7.5 vs 0.4 pg/mL; P = .0002), IL-2 (mean 5.7 vs 1.0 pg/mL; P =.0002), IL-1β (mean, 23.0 vs 11.3 pg/mL; P ≤ .0001), TNF-α (mean, 4.1 vs 1.2 pg/mL; P = .01), IL-4 (mean, 1.4 vs 0.1 pg/mL; P ≤ .0001), IL-10 (mean, 16.8 vs 7.5 pg/mL; P = .03), and IL-13 (mean, 4.5 vs 0.8 pg/mL; P ≤ .0001). Profiling cytokines in multiple sclerosis may help to identify mechanisms involved in the pathogenesis of the disease, aid in monitoring the disease course and in evaluating responses to specific therapies, and, potentially, lead to new therapies directed at cytokines or their receptors.

title: Endocrine and immune substrates of depressive symptoms and fatigue in multiple sclerosis patients with comorbid major depression
abstract: Depression and fatigue are among the most common symptoms of multiple sclerosis (MS). These symptoms frequently co-occur and partially overlap in MS but their underlying biological substrates are unclear. In this study, the relative role of cytokines and hypothalamic–pituitary–adrenal (HPA) axis activity in depression and fatigue were examined in patients with relapsing–remitting MS (RRMS).  HPA axis function and frequency of stimulated cytokine (interferon γ (IFNγ) and tumour necrosis factor α (TNFα)) producing T cells was measured cross sectionally in 44 female patients with RRMS. All subjects completed a neurological examination, the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) and self-report questionnaires.  patients met diagnostic criteria for major depressive disorder (MDD). MS patients with comorbid MDD showed normal morning but elevated evening salivary cortisol levels, resulting in a flattened slope. While a higher frequency of cytokine producing CD8+ T cells was also seen in MS patients with MDD, these markers were more closely associated with fatigue than depression.  This study supports a role for HPA axis hyperactivity in major depression in MS. In addition, inflammatory and neuroendocrine factors may differentially mediate fatigue and depressive symptoms.

title: Fatigue in multiple sclerosis: an example of cytokine mediated sickness behaviour?
abstract: Fatigue is a major complaint of multiple sclerosis (MS) patients. However, little is known about its pathophysiological mechanisms. Evidence from chronic fatigue syndrome and studies on sickness behaviour suggest that immune and neuroendocrine factors may play a causative role in the development of fatigue.  We compared whole blood stimulatory capacity for pro- (TNFα, IFNγ) and anti-inflammatory cytokines (IL-10) as well as hypothalamo-pituitary-adrenal (HPA) axis function in 15 MS patients with marked fatigue and 15 patients without fatigue as determined by the Fatigue Severity Scale (FSS).  Proinflammatory cytokines were significantly higher (TNFα: 478.9 v 228.2 pg/ml, p = 0.01; IFNγ: 57.6 v 27.8 pg/ml; p = 0.01) in MS patients with fatigue. Furthermore, TNFα values significantly correlated with daytime sleepiness as measured by the Epworth Sleepiness Scale (r = 0.64, p = 0.001). Controlling for disease activity (as measured by the Cambridge Multiple Sclerosis Basic Score), disease duration, Expanded Disability Status Scale, and depression further increased the correlation of cytokine production and fatigue. HPA axis activity was not related to fatigue but was modestly correlated with cognitive impairment.  Our data suggest that fatigue in MS is at least partially mediated through activation of proinflammatory cytokines. In line with earlier findings, HPA axis dysfunction seems not to be relevant in MS fatigue pathogenesis but appears to be linked to cognitive impairment. Our findings suggest that increased levels of inflammatory cytokines may be involved in MS fatigue. Investigation of cytokine profiles may increase the understanding of fatigue pathogenesis in MS.

title: Cytokine mRNA expression in patients with multiple sclerosis and fatigue
abstract: Fatigue is one of the most common disabling symptoms in patients with multiple sclerosis (MS), but the putative role of proinflammatory cytokines remains to be elucidated.  Thirty-seven patients (27 women, 10 men) with relapsing-remitting (n =29) and secondary progressive (n =8) MS, aged 41.0 ± 10.2 years, were studied. Fatigue was assessed by Krupp’s Fatigue Severity Scale (FSS). C ytokine mRNA expression for interferon (IFN)-g, tumor necro sis factor (TNF)-a and interleukin (IL)-10 were measured by real time RT PC R. A utonomic function was evaluated by standard tests for parasympathetic and sympathetic function, as well as by serum levels of norepinephrine and epinephrine.  Median levels of TNF-a mRNA expression were significantly higher in MS patients with (FSS]-4.0 and]-5.0, n=26 and n=14, respectively) than in those without fatigue (FSSB-4.0, n =11). No differences were seen for IFN-g and IL-10 mRNA expression. C ytokine levels were not correlated to autonomic tests or to serum catecho lamine levels.  These results suggest that TNF-a, as a principal proinflammatory mediator, is associated with MS-related fatigue. This is in support of a patho genic role of the MS-related inflammatory process in the development of fatigue.

title: Heterogeneity in Multiple Sclerosis: Scratching the Surface of a Complex Disease
abstract: Multiple Sclerosis (MS) is the most common demyelinating disease of the central nervous system. Although the etiology and the pathogenesis of MS has been extensively investigated, no single pathway, reliable biomarker, diagnostic test, or specific treatment have yet been identified for all MS patients. One of the reasons behind this failure is likely to be the wide heterogeneity observed within the MS population. The clinical course of MS is highly variable and includes several subcategories and variants. Moreover, apart from the well-established association with the HLA-class II DRB1*15:01 allele, other genetic variants have been shown to vary significantly across different populations and individuals. Finally both pathological and immunological studies suggest that different pathways may be active in different MS patients. We conclude that these “MS subtypes” should still be considered as part of the same disease but hypothesize that spatiotemporal effects of genetic and environmental agents differentially influence MS course. These considerations are extremely relevant, as outcome prediction and personalised medicine represent the central aim of modern research.

title: Distinct Patterns of Multiple Sclerosis Pathology Indicates Heterogeneity in Pathogenesis
abstract: Multiple sclerosis is an inflammatory demyeli‐nating disease of the central nervous system. The hallmark of its pathology is the demyelinated plaque with reactive glial scar formation. However, a detailed analysis of the patterns of demyelination, oligodendroglia cell pathology and the reaction of other tissue components suggests that the pathogenesis of myelin destruction in this disease may be heterogeneous. In this review we present a new classification scheme of lesional activity on the basis of the molecular composition of myelin degradation products in macrophages. When these criteria are used, different patterns of demyelination can be distinguished, including demyelination with relative preservation of oligodendrocytes, myelin destruction with concomitant and complete destruction of oligodendrocytes or primary destruction or disturbance of myelinating cells with secondary demyelination. Furthermore, in some cases a primary selective demyelination may be followed by secondary oligodendrocyte loss in the established lesions. Finally, some extraordinarily severe conditions may result in destructive lesions with loss of myelin, oligodendrocytes, axons and astro‐cytes. This heterogeneity of plaque pathology is discussed in the context of recent experimental models of inflammatory demyelination, which show that different immunological pathways may lead to the formation of demyelinated plaques that reveal the diverse structural aspects described above. Our data indicate, that the demyelinated plaques of multiple sclerosis may reflect a common pathological end point of a variety of different immunological mechanisms of myelin destruction in this disease.

title: Oxidative Stress Is Differentially Present in Multiple Sclerosis Courses, Early Evident, and Unrelated to Treatment
abstract: Oxidative stress is well documented in multiple sclerosis (MS) lesions, but its correspondence at peripheral level is still controversial.  To evaluate peripheral oxidative stress markers in MS patients.  We studied total blood levels of Coenzyme Q10 (CoQ10), oxidized and reduced forms of glutathione, malondialdehyde, reactive oxygen species (ROS), anti-oxidized-low-density lipoproteins (anti-oxLDL) antibodies, and antioxidant power (PAO) in 87 patients with different MS clinical phenotypes and in 77 controls.  CoQ10 was lower whereas anti-oxLDL antibodies titer was higher in MS patients than in controls. The benign variant of MS displayed both higher CoQ10 and higher anti-oxLDL than other MS clinical variants. Female patients had lower CoQ10 and PAO and higher ROS than male patients. Differences were greater in younger patients with shorter disease duration. Surprisingly, there was no difference for these markers between treated and untreated patients.  We found lower antioxidant agents and higher anti-oxLDL antibodies in MS, and the highest antibody titers occurred in the benign form. We suggest that natural anti-oxLDL antibodies can be protective against MS, saving blood brain barrier integrity. Our findings also suggest that milder MS is associated with a distinct oxidative stress pattern, which may provide a useful biomarker of disease prognosis.

title: Oxidative modification of patient's plasma proteins and its role in pathogenesis of multiple sclerosis.
abstract: Oxidative stress plays an important role in multiple sclerosis (MS).



The present study was designed to evaluate the modifications of plasma proteins by estimation markers of oxidative/nitrosative stress: carbonyl groups and 3-nitrotyrosines (3-NT) levels in relapsing-remitting (RR) (n=10) and secondary progressive (SP) (n=10) clinical course of multiple sclerosis. Moreover, we estimated the level of uric acid (UA) in plasma of MS patients.



Compared to controls (n=10), the levels of carbonyl groups in plasma proteins were elevated (P<0.0001) as well in RRMS as in SPMS. The highest concentration of 3-NT was observed in plasma proteins obtained from SPMS patients (P<0.0005). The level of uric acid in plasma was significantly lower in RRMS (P<0.0001) than SPMS.



This is the first report which presented differences between SPMS and RRMS patients in 3-NT and protein carbonyl groups in plasma proteins.

title: Neurodegeneration in multiple sclerosis: The role of oxidative stress and excitotoxicity
abstract: In multiple sclerosis (MS) disability results from neuronal and axonal loss, the hallmark of neurodegenerative diseases (ND). Neurodegeneration is initiated by microglia activation and mediated by oxidative stress and excitotoxicity. The same sequence of events has been consistently observed in MS. However, microglia activation correlates with a marked cell infiltration in MS but not in ND. In both pathological states, peroxynitrite is the common initiating factor of oxidative stress and excitotoxicity and is thus a potential interesting therapeutic target. Oxidative stress leads to multiple lipid and protein damages via peroxidation and nitration processes. The pathomechanisms of excitotoxicity are complex involving glutamate overload, ionic channel dysfunction, calcium overload, mitochondriopathy, proteolytic enzyme production and activation of apoptotic pathways. The inflammatory component in MS is important for the design of therapeutic strategies. Inflammation not only causes axonal and neuronal loss but it also initiates the degenerative cascade in the early stage of MS. Potent anti-inflammatory agents are now available and it is not unreasonable to think that an early blockade of inflammatory processes might also block associated degenerative mechanisms and delay disability progression. The development of neuroprotective drugs is more problematic. Indeed, given the multiple and parallel mechanisms involved in neurodegeneration, modulation of a single specific pathway will likely yield a partial benefit if any.

title: The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease.
abstract: Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C(609)T variant genotypes was observed among MS patients compared to controls (P=0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P=0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P=0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C(609)T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.

title: Elevated protein carbonylation in the brain white matter and gray matter of patients with multiple sclerosis
abstract: Oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS). Increased levels of reactive oxygen species (ROS) derived from infiltrating macrophages and microglial cells have been shown to reduce the levels of endogenous antioxidants and to cause the oxidation of various substrates within the MS plaque. To determine whether oxidative damage takes place beyond visible MS plaques, the occurrence of total carbonyls (TCOs) and protein carbonyls (PCOs) in the normal‐appearing white matter (NAWM) and gray matter (NAGM) of eight MS brains was assessed and compared with those of four control brains. The data show that most (7/8) of the MS‐WM samples contain increased amounts of PCOs as determined by reaction with 2,4‐dinitrophenylhydrazine and Western blot analysis. These samples also have high levels of glial fibrilary acidic protein (GFAP), suggesting that oxidative damage is related to the presence of small lesions. In contrast, we detected no evidence of protein thiolation (glutathionylation and cysteinylation) in the diseased tissue. To our surprise, MS‐NAGM specimens with high GFAP content also showed three times the concentration of TCOs and PCOs as the controls. The increase in PCOs is likely to be a consequence of reduced levels of antioxidants, in that the concentration of nonprotein thiols in both MS‐WM and ‐GM decreased by 30%. Overall, our data support the current view that both NAWM and ‐GM from MS brains contain considerable biochemical alterations. The involvement of GM in MS was also supported by the decrease in the levels of neurofilament light protein in all the specimens analyzed. To the best of our knowledge, this is the first study demonstrating the presence of increased protein carbonylation in post‐mortem WM and GM tissue of MS patients.

title: Cerebrospinal fluid isoprostane shows oxidative stress in patients with multiple sclerosis
abstract: The CSF level of the isoprostane 8-epi-prostaglandin (PG)-F2α (a reliable marker of oxidative stress in vivo) was three times higher in subjects with definite MS than in a benchmark group of subjects with other neurologic diseases. This increase was not correlated with that of PGE2 levels, measured as an index of cyclooxygenase activity, and was much lower in steroid-treated patients. The levels of 8-epi-PGF2α were moderately correlated with the degree of disability.

title: Evidence for increased lipid peroxidation in multiple sclerosis
abstract: Pentane and ethane are degradation products of unsaturated fatty acids which are released during lipid peroxidation. In order to assess whether multiple sclerosis is associated with lipid peroxidation, we measured pentane and ethane excretion by 16 patients with multiple sclerosis and compared them to healthy control subjects. Patients with acute exacerbation of multiple sclerosis had significantly higher concentrations of pentane (10.5±4.2 nmol/l)(p<0.01) compared to either patients in remission (4.5±1.7 nmol/l) or control subjects (4.9±1.1 nmol/l). The concentrations of ethane were not significantly different among these groups. Of the patients with acute exacerbation who later achieved remission, the pentane excretion also returned to normal (5.6±0.9 nmol/l). One patient who failed to reachieve clinical remission continued to excrete large amounts of pentane. We conclude that oxygen free radical activity is enhanced during exacerbation multiple sclerosis.

title: Nitric oxide synthase is present in the cerebrospinal fluid of patients with active multiple sclerosis and is associated with increases in cerebrospinal fluid protein nitrotyrosine and S‐nitrosothiols and with changes in glutathione levels
abstract: Nitric oxide (NO) is hypothesized to play a role in the immunopathogenesis of multiple sclerosis (MS). Increased levels of NO metabolites have been found in patients with MS. Peroxynitrite, generated by the reaction of NO with superoxide at sites of inflammation, is a strong oxidant capable of damaging tissues and cells. Inducible NO synthase (iNOS) is up‐regulated in the CNS of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. In this study, Western blots of cerebrospinal fluid (CSF) from patients with MS demonstrated the presence of iNOS, which was absent in CSF from control subjects. There was also NOS activity present in both MS and control CSF. Total NOS activity was increased (by 24%) in the CSF from MS patients compared with matched controls. The addition of 0.1 mM ITU (a specific iNOS inhibitor) to the samples did not change the activity of the control samples but decreased the NOS activity in the MS samples to almost control levels. The addition of 1 mM L‐NMMA (a nonisoform specific NOS inhibitor), completely inhibited NOS activity in CSF from control and MS subjects. Nitrotyrosine immunostaining of CSF proteins was detectable in controls but was greatly increased in MS samples. There were also significant increases in CSF nitrate + nitrite and oxidant‐enhanced luminescence in MS samples compared with controls. Additionally, a significant decrease in reduced glutathione and significant increases in oxidized glutathione and S‐nitrosothiols were found in MS samples compared with controls. Parallel changes in NO metabolites were observed in the plasma of MS patients, compared with controls, and accompanied a significant increase of reduced glutathione. These data strongly support a role for nitrosative stress in the pathogenesis of MS and indicate that therapeutic strategies focussed on decreasing production of NO by iNOS and/or scavenging peroxynitrite may be useful in alleviating the neurological impairments that occur during MS relapse.

title: Elevated cerebrospinal fluid F2-isoprostane levels indicating oxidative stress in healthy siblings of multiple sclerosis patients
abstract: Lipid peroxidation has been implicated in the pathogenesis of multiple sclerosis (MS). Isoprostanes, isomers of prostaglandins, are produced by free radical-mediated peroxidation of fatty acids in vivo and can be quantified in biological fluids. This study examines the levels of cerebrospinal fluid (CSF) F2-isoprostanes (F2-iPs) in MS patients (n=46), their healthy siblings (n=46) and unrelated controls (n=50). The median CSF F2-iP concentration (range) was significantly higher in siblings of MS patients, as compared to healthy controls (40.0 [7.1-68.7] and 29.1 [6.4-60.3] pg/mL, respectively, p=0.031). MS patients demonstrated F2-iP levels intermediate between siblings and controls. F2-iP levels in MS patients and siblings correlated significantly (R=0.360, p=0.012). These results suggest that siblings of MS patients have an increased oxidative stress response to environmental and/or genetic factors that may be involved in MS pathogenesis.

title: Contrasting potential of nitric oxide and peroxynitrite to mediate oligodendrocyte injury in multiple sclerosis
abstract: Nitric oxide (NO) and peroxynitrite (ONOO−) are potential mediators of the injury and cytotoxicity occurring over time to oligodendrocytes in multiple sclerosis (MS) lesions. Our in vitro results indicate that human adult CNS‐derived oligodendrocytes are relatively resistant to NO‐mediated damage. In contrast, human oligodendrocytes are highly susceptible to peroxynitrite‐mediated injury. In situ, we found that inducible nitric oxide synthase (iNOS) was expressed in astrocytes and macrophages in all active demyelinating and remyelinating MS lesions examined, yet no correlation was found between numbers of glial cells expressing iNOS and the extent of oligodendrocyte cell death. Nitrotyrosine groups, indicative of the presence of peroxynitrite in vivo, could be detected on astrocytes, macrophages, and oligodendrocytes in MS lesions. High numbers of nitrotyrosine‐positive oligodendrocytes were found in one MS case that featured extensive oligodendrocyte cell death. Our results indicate that NO alone is unlikely to induce oligodendrocyte injury, whereas its more potent byproduct peroxynitrite is a potential mediator of injury to oligodendrocytes in MS.

title: Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis.
abstract: Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) disease states, including the animal model of multiple sclerosis (MS) and experimental allergic encephalomyelitis. We have examined post-mortem brain tissues collected from patients previously diagnosed with MS, as well as tissues collected from the brains of patients dying without neuropathies. Both Northern blot analysis and reverse transcriptase (RT)-driven in situ PCR (RT-in situ PCR) studies demonstrated that inducible NO synthase (iNOS) mRNA was present in the brain tissues from MS patients but was absent in equivalent tissues from normal controls. We have also performed experiments identifying the cell type responsible for iNOS expression by RT-in situ PCR in combination with immunohistochemistry. Concomitantly, we analyzed the tissues for the presence of the NO reaction product nitrotyrosine to demonstrate the presence of a protein nitrosylation adduct. We report here that iNOS mRNA was detectable in the brains of 100% of the CNS tissues from seven MS patients examined but in none of the three normal brains. RT-in situ PCR experiments also demonstrated the presence of iNOS mRNA in the cytoplasm of cells that also expressed the ligand recognized by the Ricinus communis agglutinin 1 (RCA-1), a monocyte/macrophage lineage marker. Additionally, specific labeling of cells was observed when brain tissues from MS patients were exposed to antisera reactive with nitrotyrosine residues but was significantly less plentiful in brain tissue from patients without CNS disease. These results demonstrate that iNOS, one of the enzymes responsible for the production of NO, is expressed at significant levels in the brains of patients with MS and may contribute to the pathology associated with the disease.

title: The potential role of nitric oxide in multiple sclerosis
abstract: Nitric oxide (.NO) and its reactive derivative peroxynitrite (ONOO7) have been implicated in the pathogenesis of multiple sclerosis (MS). They are cytotoxic to oligodendrocytes and neurones in culture by inhibiting the mitochondrial respiratory chain (complexes II/III and IV) and inhibiting certain key intracellular enzymes. Recently .NO has been implicated as a possible aetiological factor in reversible conduction block in demyelinated axons. Inducible nitric oxide synthase (iNOS) is upregulated in the central nervous system of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. In some EAE models inhibiting iNOS activity decreases disease severity whilst in other models disease activity is exacerbated. Raised levels of nitrate and nitrite, stable end-products of .NO/ONOO7, are found in the cerebrospinal fluid, serum and urine of patients with MS. CSF levels of nitrate and nitrite correlate with blood-brain-barrier dysfunction, which suggests that .NO may play a role in inflammatory blood-brain-barrier dysfunction. In a longitudinal study on 24 patients with relapsing remitting and secondary progressive MS, raised serum nitrate and nitrite levels correlated with a relapsing course and infrequent relapses. However, no correlation was found between raised serum levels of nitrate and nitrite and MRI activity, disease progression, or the development of cerebral atrophy. In autoimmune mediated CNS demyelinating disease .NO may be a double-edged sword, mediating tissue damage on the one hand and on the other hand modulating complex immunological functions which may be protective.

title: Effect of natalizumab on oxidative damage biomarkers in relapsing-remitting multiple sclerosis.
abstract: Natalizumab is a monoclonal antibody used to treat multiple sclerosis. This study sought to determine whether the protective action of natalizumab involved a reduction in oxidative damage.



Twenty-two multiple sclerosis patients fulfilling the revised McDonald criteria were assigned to treatment with 300 mg natalizumab intravenously once monthly (infusion every 4 weeks) in accordance with Spanish guidelines. Carbonylated proteins, 8-hydroxy-2'-deoxyguanosine, total glutathione, reduced glutathione, superoxide dismutase, glutathione peroxidase, and myeloperoxidase levels were measured at baseline and after 14 months' treatment, and the antioxidant gap was calculated.



Natalizumab prompted a drop in oxidative-damage biomarker levels, together with a reduction both in myeloperoxidase levels and in the myeloperoxidase/neutrophil granulocyte ratio. Interestingly, natalizumab induced nuclear translocation of Nrf2 and a fall in serum vascular cell adhesion molecule-1 levels.



These findings suggest that natalizumab has a beneficial effect on oxidative damage found in MS patients.

title: Involvement of Oxidative Stress in Occurrence of Relapses in Multiple Sclerosis: The Spectrum of Oxidatively Modified Serum Proteins Detected by Proteomics and Redox Proteomics Analysis
abstract: Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Several evidences suggest that MS can be considered a multi-factorial disease in which both genetics and environmental factors are involved. Among proposed candidates, growing results support the involvement of oxidative stress (OS) in MS pathology. The aim of this study was to investigate the role of OS in event of exacerbations in MS on serum of relapsing-remitting (RR-MS) patients, either in relapsing or remitting phase, with respect to serum from healthy subjects. We applied proteomics and redox proteomics approaches to identify differently expressed and oxidatively modified proteins in the low-abundant serum protein fraction. Among differently expressed proteins ceruloplasmin, antithrombin III, clusterin, apolipoprotein E, and complement C3, were up-regulated in MS patients compared with healthy controls. Further by redox proteomics, vitamin D-binding protein showed a progressive trend of oxidation from remission to relapse, respect with controls. Similarly, the increase of oxidation of apolipoprotein A-IV confirmed that levels of OS are elevated with the progression of the disease. Our findings support the involvement of OS in MS and suggest that dysfunction of target proteins occurs upon oxidative damage and correlates with the pathology.

title: Cerebrospinal fluid brain specific proteins in relation to nitric oxide metabolites during relapse of multiple sclerosis
abstract: This study investigated the cerebrospinal fluid (CSF) levels of ferritin, S100B as biomarkers for glial activation and NfHSM135 — a biomarker of axonal damage — in relation to nitric oxide (NO) metabolites: nitrate and nitrite (NOx) during acute multiple sclerosis (MS) relapse. Thirty-four relapsing—remitting MS (RR-MS) patients during acute relapse and 12 controls were enrolled. Patients were assessed on Expanded Disability Status Scale (EDSS) and underwent lumbar puncture within two weeks following relapse. Twenty patients were available for further follow-up and were assessed on EDSS 6—8 weeks since the relapse onset. The CSF NOx (P < 0.0001), NfHSM135 ( P = 0.01) and S100B (P = 0.009) but not ferritin (P > 0.05) were significantly raised in MS group. There was a significant correlation between CSF ferritin and S100B in RR-MS group (P = 0.004). CSF NOx did not correlate with S100B and ferritin in study groups. RR-MS patients with detectable NfHSM135 levels had higher NOx compared with subjects having undetectable NfHSM135 (P = 0.03). In the follow-up study, raised baseline levels of NOx (P = 0.016) or NfHSM135 (P = 0.04) inversely correlated with the clinical recovery grade expressed as relative EDSS change between baseline and follow-up. In conclusion, NO metabolites were increased and because of their correlation with a biomarker of axonal degeneration (neurofilaments) and a measure for clinical disability (EDSS), relapse-related nitrosative stress is likely to be relevant to the development of sustained disability in an individual patient. Multiple Sclerosis 2008; 14: 59—66.

title: Clonal expansion of mitochondrial DNA deletions and the progression of multiple sclerosis.
abstract: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Mechanisms of disease progression in MS are poorly understood but are thought to relate to both focal pathology as well as diffuse inflammation in the white and grey matter. Evidence points to neurodegeneration combined with a loss of cellular function in the remaining tissue as an important factor to the progression of MS. Mitochondria are implicated to play a role in the pathogenesis of MS with evidence of loss of mitochondrial respiratory chain activity, down regulation of both nuclear DNA and mitochondrial DNA (mtDNA) encoded transcripts as well as oxidative damage to, and deletions of, the mitochondrial DNA (mtDNA). The double stranded circle of mtDNA (16.6 kb) encompasses genes encoding key subunits within the mitochondrial respiratory chain required for the production of ATP as well as transfer RNA and ribosomal RNA molecules within the cell. The stability of mtDNA is essential for a healthy CNS as highlighted by the patients with primary mitochondrial disease. In this review, we focus on the potential role of mtDNA mutations, in particular somatic mtDNA deletions, in the pathogenesis of the progressive stage of MS. We propose clonal expansion of somatic mtDNA deletions as a potential molecular link between early inflammatory events and a delayed cellular energy failure, dysfunction and degeneration. The high level of somatic mtDNA deletions within single cells in MS is likely to cause cellular dysfunction as well as increase the susceptibility of the CNS tissue to additional stress.

title: The link between inflammation, synaptic transmission and neurodegeneration in multiple sclerosis
abstract: Multiple sclerosis (MS) has been classically regarded as a disorder of the white matter of the central nervous system (CNS). However, early alterations of the neuronal compartment occurring in this disorder are partially independent of demyelination. Soluble inflammatory cytokines and glutamate have been proposed as major determinants of neurodegeneration in MS as well as in its experimental animal model, namely experimental autoimmune encephalomyelitis (EAE). The relationship between these two major determinants has been largely elusive. In recent years, unexpected connections have emerged between immune cells and soluble cytokines on the one hand, and synaptic transmission and neurodegeneration on the other. Neurophysiological recordings have recently shown that glutamate-mediated excitatory postsynaptic currents are enhanced during the early phase of EAE, because of altered expression and phosphorylation of AMPA receptors and the downregulation of the immediate early gene Arc/Arg3.1. The synaptic alterations occurring during neuroinflammatory diseases are largely mediated by inflammatory cytokines released from infiltrating T cells and from activated microglia, and are responsible, at least in part, for irreversible dendritic pathology. Collectively, the data covered in this review article suggest that CNS-confined inflammation in MS is associated with the release of soluble molecules, which are capable of altering excitatory synaptic transmission and, finally, of stimulating secondary neurodegenerative gray matter pathology.

title: Neurodegeneration from mitochondrial insufficiency: nutrients, stem cells, growth factors, and prospects for brain rebuilding using integrative management.
abstract: Degenerative brain disorders (neurodegeneration) can be frustrating for both conventional and alternative practitioners. A more comprehensive, integrative approach is urgently needed. One emerging focus for intervention is brain energetics. Specifically, mitochondrial insufficiency contributes to the etiopathology of many such disorders. Electron leakages inherent to mitochondrial energetics generate reactive oxygen free radical species that may place the ultimate limit on lifespan. Exogenous toxins, such as mercury and other environmental contaminants, exacerbate mitochondrial electron leakage, hastening their demise and that of their host cells. Studies of the brain in Alzheimer's and other dementias, Down syndrome, stroke, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, Friedreich's ataxia, aging, and constitutive disorders demonstrate impairments of the mitochondrial citric acid cycle and oxidative phosphorylation (OXPHOS) enzymes. Imaging or metabolic assays frequently reveal energetic insufficiency and depleted energy reserve in brain tissue in situ. Orthomolecular nutrients involved in mitochondrial metabolism provide clinical benefit. Among these are the essential minerals and the B vitamin group; vitamins E and K; and the antioxidant and energetic cofactors alpha-lipoic acid (ALA), ubiquinone (coenzyme Q10; CoQ10), and nicotinamide adenine dinucleotide, reduced (NADH). Recent advances in the area of stem cells and growth factors encourage optimism regarding brain regeneration. The trophic nutrients acetyl L-carnitine (ALCAR), glycerophosphocholine (GPC), and phosphatidylserine (PS) provide mitochondrial support and conserve growth factor receptors; all three improved cognition in double-blind trials. The omega-3 fatty acid docosahexaenoic acid (DHA) is enzymatically combined with GPC and PS to form membrane phospholipids for nerve cell expansion. Practical recommendations are presented for integrating these safe and well-tolerated orthomolecular nutrients into a comprehensive dietary supplementation program for brain vitality and productive lifespan.

title: Reduced expression of PGC-1α partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex
abstract: There is growing evidence that mitochondrial dysfunction and associated reactive oxygen species (ROS) formation contribute to neurodegenerative processes in multiple sclerosis (MS). Here, we investigated whether alterations in transcriptional regulators of key mitochondrial proteins underlie mitochondrial dysfunction in MS cortex and contribute to neuronal loss. Hereto, we analyzed the expression of mitochondrial transcriptional (co-)factors and proteins involved in mitochondrial redox balance regulation in normal-appearing grey matter (NAGM) samples of cingulate gyrus and/or frontal cortex from 15 MS patients and nine controls matched for age, gender and post-mortem interval. PGC-1α, a transcriptional co-activator and master regulator of mitochondrial function, was consistently and significantly decreased in pyramidal neurons in the deeper layers of MS cortex. Reduced PGC-1α levels coincided with reduced expression of oxidative phosphorylation subunits and a decrease in gene and protein expression of various mitochondrial antioxidants and uncoupling proteins (UCPs) 4 and 5. Short-hairpin RNA-mediated silencing of PGC-1α in a neuronal cell line confirmed that reduced levels of PGC-1α resulted in a decrease in transcription of OxPhos subunits, mitochondrial antioxidants and UCPs. Moreover, PGC-1α silencing resulted in a decreased mitochondrial membrane potential, increased ROS formation and enhanced susceptibility to ROS-induced cell death. Importantly, we found extensive neuronal loss in NAGM from cingulate gyrus and frontal cortex of MS patients, which significantly correlated with the extent of PGC-1α decrease. Taken together, our data indicate that reduced neuronal PGC-1α expression in MS cortex partly underlies mitochondrial dysfunction in MS grey matter and thereby contributes to neurodegeneration in MS cortex.

title: Variations in Mitochondrial DNA Copy Numbers in MS Brains
abstract: The aim of this study is to determine if there is a pathology-related variation in mitochondrial (mt)DNA copy numbers in brains of patients with multiple sclerosis (MS). Our recent study demonstrated an age-dependent but excluded a MS pathology-related increase in the proportion of cytochrome oxidase (COX)-negative cells and deleted mtDNA molecules in postmortem brain tissue specimens of patients and controls (Blokhin et al., Neuromolecular Medicine,  2008). This corollary study further extends our efforts defining mitochondrial contributions to tissue degeneration associated with inflammatory demyelination. Copy number variations of mtDNA molecules were defined by quantifying the mtDNA ND1 gene copies relative to the invariable nuclear ribosomal 18S gene copies (ND1/r18S) using real-time polymerase chain reaction analyses in laser dissected, COX-positive and COX-negative single neurons and glial cells from frozen postmortem normal-appearing gray (NAGM) and white matter (NAWM) regions and chronic active plaques of MS patients, and gray matter (GM) and white matter (WM) regions of age matched non-neurological disease (NND) controls. ND1/r18S values were correlated with tissue regions, pathology, and age. While the ND1/r18S values were similar in NAWM and plaque-containing specimens of MS patients as well as in NAWM of patients and WM of age-matched NND controls, we found significantly higher mtDNA copy number values in neurons of NAGM than in cells of other MS brain regions. The ND1/r18S values were even higher in NAGM than in GM of age-matched NND controls. An age-related decline in ND1/r18S values was also noted in neurons of both MS patients and NND controls. These observations exclude a change in mtDNA copy numbers in plaques, however, suggest a compensatory replication of mtDNA or mitochondria in the cortex with neuroaxonal loss in MS. The age-related decline in mtDNA copy numbers may explain some features of late-onset MS.

title: Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis
abstract: Soluble products of activated immune cells include reactive oxygen species (ROS) and nitric oxide (NO) with a high potential to induce biochemical modifications and degenerative changes in areas of inflammation in the central nervous system (CNS). Previously, we demonstrated an increased production of ROS by activated mononuclear cells (MNC) of patients with multiple sclerosis (MS) compared to those of controls, and development of oxidative damage to total DNA in association with inflammation in chronic active plaques. The current study aimed to determine whether mitochondrial (mt)DNA is affected by oxidative damage, and whether oxidative damage to mitochondrial macromolecules (including mtDNA) is associated with a decline in the activity of mitochondrial enzyme complexes. Using molecular and biochemical methods we demonstrate a trend for impaired NADH dehydrogenase (DH) activity and a possible compensatory increase in complex IV activity in association with oxidative damage to mtDNA in chronic active plaques. Immunohistochemistry confirms the increase of oxidative damage to DNA predominantly located in the cytoplasmic compartment of cells in chronic active plaques. These observations suggest that oxidative damage to macromolecules develops in association with inflammation in the CNS, and may contribute to a decline of energy metabolism in affected cells. As observed in neurodegenerative diseases of non-inflammatory origin, decreased ATP synthesis can ultimately lead to cell death or degeneration. Therefore, elucidation of this pathway in MS deserves further studies which may identify neuroprotective strategies to prevent tissue degeneration and the associated clinical disability.

title: Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients
abstract: Degeneration of chronically demyelinated axons is a major cause of irreversible neurological disability in multiple sclerosis (MS) patients. Development of neuroprotective therapies will require elucidation of the molecular mechanisms by which neurons and axons degenerate.

title: Mitochondrial changes within axons in multiple sclerosis.
abstract: Multiple sclerosis is the most common cause of non-traumatic neurological impairment in young adults. An energy deficient state has been implicated in the degeneration of axons, the pathological correlate of disease progression, in multiple sclerosis. Mitochondria are the most efficient producers of energy and play an important role in calcium homeostasis. We analysed the density and function of mitochondria using immunohistochemistry and histochemistry, respectively, in chronic active and inactive lesions in progressive multiple sclerosis. As shown before in acute pattern III and Balo's lesions, the mitochondrial respiratory chain complex IV activity is reduced despite the presence of mitochondria in demyelinated axons with amyloid precursor protein accumulation, which are predominantly located at the active edge of chronic active lesions. Furthermore, the strong non-phosphorylated neurofilament (SMI32) reactivity was associated with a significant reduction in complex IV activity and mitochondria within demyelinated axons. The complex IV defect associated with axonal injury may be mediated by soluble products of innate immunity, as suggested by an inverse correlation between complex IV activity and macrophage/microglial density in chronic lesions. However, in inactive areas of chronic multiple sclerosis lesions the mitochondrial respiratory chain complex IV activity and mitochondrial mass, judged by porin immunoreactivity, are increased within approximately half of large (>2.5 microm diameter) chronically demyelinated axons compared with large myelinated axons in the brain and spinal cord. The axon-specific mitochondrial docking protein (syntaphilin) and phosphorylated neurofilament-H were increased in chronic lesions. The lack of complex IV activity in a proportion of Na(+)/K(+) ATPase alpha-1 positive demyelinated axons supports axonal dysfunction as a contributor to neurological impairment and disease progression. Furthermore, in vitro studies show that inhibition of complex IV augments glutamate-mediated axonal injury (amyloid precursor protein and SMI32 reactivity). Our findings have important implications for both axonal degeneration and dysfunction during the progressive stage of multiple sclerosis.

title: Metabolomic profiling in multiple sclerosis: insights into biomarkers and pathogenesis
abstract: Metabolomics enables the provision of sensitive bio-markers of disease. We performed 800 MHz 1H-nuclear magnetic resonance (NMR) spectroscopic analyses of cerebrospinal fluid (CSF) specimens to identify biomarkers of multiple sclerosis (MS), yielding reproducible detection of 15 metabolites from MS (n=15) and non-MS (n=17) patients. Mean levels of choline, myo-inositol and threonate were increased, whereas 3-hydroxybutyrate, citrate, phenylalanine, 2-hydroxyisovalerate and mannose were decreased in MS-derived CSF (p<0.05), suggesting alterations to energy and phospholipid metabolism. Multivariate hierarchal cluster analysis indicated a high correlation within the metabolite profiles, significantly clustering samples into the two clinical groups, which was corroborated using principal components analysis. CSF metabolomics have the capacity to yield quantitative biomarkers and insights into the pathogenesis of MS.

title: Cerebrospinal fluid ATP metabolites in multiple sclerosis
abstract: Increased axonal energy demand and mitochondrial failure have been suggested as possible causes for axonal degeneration and disability in multiple sclerosis. Our objective was to test whether ATP depletion precedes clinical, imaging and biomarker evidence for axonal degeneration in multiple sclerosis. The method consisted of a longitudinal study which included 21 patients with multiple sclerosis. High performance liquid chromatography was used to quantify biomarkers of the ATP metabolism (oxypurines and purines) from the cerebrospinal fluid at baseline.  were quantified at baseline and 3-year follow-up. Central ATP depletion (sum of ATP metabolites >19.7 µmol/litre) was followed by more severe progression of disability if compared to normal ATP metabolites (median 1.5 versus 0, p< 0.05). Baseline ATP metabolite levels correlated with change of Expanded Disability Status Scale in the pooled cohort (r= 0.66, p= 0.001) and subgroups (relapsing—remitting patients: r= 0.79, p< 0.05 and secondary progressive/primary progressive patients: r= 0.69, p< 0.01). There was no relationship between central ATP metabolites and either biomarker or MRI evidence for axonal degeneration. The data suggests that an increased energy demand in multiple sclerosis may cause a quantifiable degree of central ATP depletion. We speculate that the observed clinical disability may be related to depolarisation associated conduction block.

title: Disentangling the heterogeneity of autism spectrum disorder through genetic findings
abstract: Autism spectrum disorder (ASD) represents a heterogeneous group of disorders, which presents a substantial challenge to diagnosis and treatment. Over the past decade, considerable progress has been made in the identification of genetic risk factors for ASD that define specific mechanisms and pathways underlying the associated behavioural deficits. In this Review, we discuss how some of the latest advances in the genetics of ASD have facilitated parsing of the phenotypic heterogeneity of this disorder. We argue that only through such advances will we begin to define endophenotypes that can benefit from targeted, hypothesis-driven treatments. We review the latest technologies used to identify and characterize the genetics underlying ASD and then consider three themes—single-gene disorders, the gender bias in ASD, and the genetics of neurological comorbidities—that highlight ways in which we can use genetics to define the many phenotypes within the autism spectrum. We also present current clinical guidelines for genetic testing in ASD and their implications for prognosis and treatment.

title: Etiological heterogeneity in autism spectrum disorders: More than 100 genetic and genomic disorders and still counting
abstract: There is increasing evidence that autism spectrum disorders (ASDs) can arise from rare highly penetrant mutations and genomic imbalances. The rare nature of these variants, and the often differing orbits of clinical and research geneticists, can make it difficult to fully appreciate the extent to which we have made progress in understanding the genetic etiology of autism. In fact, there is a persistent view in the autism research community that there are only a modest number of autism loci known. We carried out an exhaustive review of the clinical genetics and research genetics literature in an attempt to collate all genes and recurrent genomic imbalances that have been implicated in the etiology of ASD. We provide data on 103 disease genes and 44 genomic loci reported in subjects with ASD or autistic behavior. These genes and loci have all been causally implicated in intellectual disability, indicating that these two neurodevelopmental disorders share common genetic bases. A genetic overlap between ASD and epilepsy is also apparent in many cases. Taken together, these findings clearly show that autism is not a single clinical entity but a behavioral manifestation of tens or perhaps hundreds of genetic and genomic disorders. Increased recognition of the etiological heterogeneity of ASD will greatly expand the number of target genes for neurobiological investigations and thereby provide additional avenues for the development of pathway-based pharmacotherapy. Finally, the data provide strong support for high-resolution DNA microarrays as well as whole-exome and whole-genome sequencing as critical approaches for identifying the genetic causes of ASDs.

title: Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits
abstract: Autism spectrum disorder (ASD) defines a group of common, complex neurodevelopmental disorders. Although the aetiology of ASD has a strong genetic component, there is considerable monozygotic (MZ) twin discordance indicating a role for non-genetic factors. Because MZ twins share an identical DNA sequence, disease-discordant MZ twin pairs provide an ideal model for examining the contribution of environmentally driven epigenetic factors in disease. We performed a genome-wide analysis of DNA methylation in a sample of 50 MZ twin pairs (100 individuals) sampled from a representative population cohort that included twins discordant and concordant for ASD, ASD-associated traits and no autistic phenotype. Within-twin and between-group analyses identified numerous differentially methylated regions associated with ASD. In addition, we report significant correlations between DNA methylation and quantitatively measured autistic trait scores across our sample cohort. This study represents the first systematic epigenomic analyses of MZ twins discordant for ASD and implicates a role for altered DNA methylation in autism.

title: Genetic heritability and shared environmental factors among twin pairs with autism.
abstract: Autism is considered the most heritable of neurodevelopmental disorders, mainly because of the large difference in concordance rates between monozygotic and dizygotic twins.



To provide rigorous quantitative estimates of genetic heritability of autism and the effects of shared environment.



Twin pairs with at least 1 twin with an autism spectrum disorder (ASD) born between 1987 and 2004 were identified through the California Department of Developmental Services.



Structured diagnostic assessments (Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule) were completed on 192 twin pairs. Concordance rates were calculated and parametric models were fitted for 2 definitions, 1 narrow (strict autism) and 1 broad (ASD).



For strict autism, probandwise concordance for male twins was 0.58 for 40 monozygotic pairs (95% confidence interval [CI], 0.42-0.74) and 0.21 for 31 dizygotic pairs (95% CI, 0.09-0.43); for female twins, the concordance was 0.60 for 7 monozygotic pairs (95% CI, 0.28-0.90) and 0.27 for 10 dizygotic pairs (95% CI, 0.09-0.69). For ASD, the probandwise concordance for male twins was 0.77 for 45 monozygotic pairs (95% CI, 0.65-0.86) and 0.31 for 45 dizygotic pairs (95% CI, 0.16-0.46); for female twins, the concordance was 0.50 for 9 monozygotic pairs (95% CI, 0.16-0.84) and 0.36 for 13 dizygotic pairs (95% CI, 0.11-0.60). A large proportion of the variance in liability can be explained by shared environmental factors (55%; 95% CI, 9%-81% for autism and 58%; 95% CI, 30%-80% for ASD) in addition to moderate genetic heritability (37%; 95% CI, 8%-84% for autism and 38%; 95% CI, 14%-67% for ASD).



Susceptibility to ASD has moderate genetic heritability and a substantial shared twin environmental component.

title: Autism spectrum disorders and autistic traits: A decade of new twin studies
abstract: Researchers continue to pursue a better understanding of the symptoms, comorbidities, and causes of autism spectrum disorders. In this article we review more than 30 twin studies of autism spectrum disorders (ASDs) and autistic traits published in the last decade that have contributed to this endeavor. These twin studies have reported on the heritability of autism spectrum disorders and autistic traits in different populations and using different measurement and age groups. These studies have also stimulated debate and new hypotheses regarding why ASDs show substantial symptom heterogeneity, and what causes their comorbidity with intellectual disability, language delay, and other psychiatric disorders such as ADHD. These studies also reveal that the etiology of autism and autistic traits assessed in the general population is more similar than different, which contributes to the question of where the boundary lies between autism and typical development. Recent findings regarding molecular genetic and environmental causes of autism are discussed in the relation to these twin studies. Lastly, methodological assumptions of the twin design are given consideration, as well as issues of measurement. Future research directions are suggested to ensure that this decade is as productive as the last in attempting to disentangle the causes of autism spectrum disorders.

title: Elevated immune response in the brain of autistic patients
abstract: This study determined immune activities in the brain of ASD patients and matched normal subjects by examining cytokines in the brain tissue. Our results showed that proinflammatory cytokines (TNF-alpha, IL-6 and GM-CSF), Th1 cytokine (IFN-gamma) and chemokine (IL-8) were significantly increased in the brains of ASD patients compared with the controls. However the Th2 cytokines (IL-4, IL-5 and IL-10) showed no significant difference. The Th1/Th2 ratio was also significantly increased in ASD patients.



ASD patients displayed an increased innate and adaptive immune response through the Th1 pathway, suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of ASD.

title: Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism
abstract: In the neurodevelopmental disorder autism, several neuroimmune abnormalities have been reported. However, it is unknown whether microglial somal volume or density are altered in the cortex and whether any alteration is associated with age or other potential covariates.



Microglia in sections from the dorsolateral prefrontal cortex of nonmacrencephalic male cases with autism (n = 13) and control cases (n = 9) were visualized via ionized calcium binding adapter molecule 1 immunohistochemistry. In addition to a neuropathological assessment, microglial cell density was stereologically estimated via optical fractionator and average somal volume was quantified via isotropic nucleator.



Microglia appeared markedly activated in 5 of 13 cases with autism, including 2 of 3 under age 6, and marginally activated in an additional 4 of 13 cases. Morphological alterations included somal enlargement, process retraction and thickening, and extension of filopodia from processes. Average microglial somal volume was significantly increased in white matter (p = .013), with a trend in gray matter (p = .098). Microglial cell density was increased in gray matter (p = .002). Seizure history did not influence any activation measure.



The activation profile described represents a neuropathological alteration in a sizeable fraction of cases with autism. Given its early presence, microglial activation may play a central role in the pathogenesis of autism in a substantial proportion of patients. Alternatively, activation may represent a response of the innate neuroimmune system to synaptic, neuronal, or neuronal network disturbances, or reflect genetic and/or environmental abnormalities impacting multiple cellular populations.

title: Autoantibodies to cerebellum in children with autism associate with behavior
abstract: Autism is a heterogeneous disorder with a poorly understood biological basis. Some children with autism harbor plasma autoantibodies that target brain proteins. Similarly, some mothers of children with autism produce antibodies specific to autism that target pairs of fetal brain proteins at 37/73 and 39/73 kDa. We explored the relationship between the presence of brain-specific autoantibodies and several behavioral characteristics of autism in 277 children with an autism spectrum disorder and 189 typically developing age-matched controls. Further, we used maternal autoantibody data to investigate potential familial relationships for the production of brain-directed autoantibodies. We demonstrated by Western blot that autoantibodies specific for a 45 kDa cerebellar protein in children were associated with a diagnosis of autism (p=0.017) while autoantibodies directed towards a 62 kDa protein were associated with the broader diagnosis of autism spectrum disorder (ASD) (p=0.043). Children with such autoantibodies had lower adaptive (p=0.0008) and cognitive function (p=0.005), as well as increased aberrant behaviors (p<0.05) compared to children without these antibodies. No correlation was noted for those mothers with the most specific pattern of anti-fetal brain autoantibodies and children with the autoantibodies to either the 45 or 62 kDa bands. Collectively, these data suggest that antibodies towards brain proteins in children are associated with lower adaptive and cognitive function as well as core behaviors associated with autism. It is unclear whether these antibodies have direct pathologic significance, or if they are merely a response to previous injury. Future studies are needed to determine the identities of the protein targets and explore their significance in autism.

title: The role of immune dysfunction in the pathophysiology of autism
abstract: Autism spectrum disorders (ASD) are a complex group of neurodevelopmental disorders encompassing impairments in communication, social interactions and restricted stereotypical behaviors. Although a link between altered immune responses and ASD was first recognized nearly 40 years ago, only recently has new evidence started to shed light on the complex multifaceted relationship between immune dysfunction and behavior in ASD. Neurobiological research in ASD has highlighted pathways involved in neural development, synapse plasticity, structural brain abnormalities, cognition and behavior. At the same time, several lines of evidence point to altered immune dysfunction in ASD that directly impacts some or all these neurological processes. Extensive alterations in immune function have now been described in both children and adults with ASD, including ongoing inflammation in brain specimens, elevated pro-inflammatory cytokine profiles in the CSF and blood, increased presence of brain-specific auto-antibodies and altered immune cell function. Furthermore, these dysfunctional immune responses are associated with increased impairments in behaviors characteristic of core features of ASD, in particular, deficits in social interactions and communication. This accumulating evidence suggests that immune processes play a key role in the pathophysiology of ASD. This review will discuss the current state of our knowledge of immune dysfunction in ASD, how these findings may impact on underlying neuro-immune mechanisms and implicate potential areas where the manipulation of the immune response could have an impact on behavior and immunity in ASD.

title: Macrophage Migration Inhibitory Factor and Autism Spectrum Disorders
abstract: Autistic spectrum disorders are childhood neurodevelopmental disorders characterized by social and communicative impairment and repetitive and stereotypical behavior. Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity that promotes monocyte/macrophage-activation responses by increasing the expression of Toll-like receptors and inhibiting activation-induced apoptosis. On the basis of results of previous genetic linkage studies and reported altered innate immune response in autism spectrum disorder, we hypothesized that MIF could represent a candidate gene for autism spectrum disorder or its diagnostic components.  Genetic association between autism spectrum disorder and MIF was investigated in 2 independent sets of families of probands with autism spectrum disorder, from the United States (527 participants from 152 families) and Holland (532 participants from 183 families). Probands and their siblings, when available, were evaluated with clinical instruments used for autism spectrum disorder diagnoses. Genotyping was performed for 2 polymorphisms in the promoter region of the MIF gene in both samples sequentially. In addition, MIF plasma analyses were conducted in a subset of Dutch patients from whom plasma was available.  There were genetic associations between known functional polymorphisms in the promoter for MIF and autism spectrum disorder–related behaviors. Also, probands with autism spectrum disorder exhibited higher circulating MIF levels than did their unaffected siblings, and plasma MIF concentrations correlated with the severity of multiple autism spectrum disorder symptoms.  These results identify MIF as a possible susceptibility gene for autism spectrum disorder. Additional research is warranted on the precise relationship between MIF and the behavioral components of autism spectrum disorder, the mechanism by which MIF contributes to autism spectrum disorder pathogenesis, and the clinical use of MIF genotyping.

title: Transcriptomic Analysis of Autistic Brain Reveals Convergent Molecular Pathology
abstract: Autism spectrum disorder (ASD) is a common, highly heritable neurodevelopmental condition characterized by marked genetic heterogeneity. Thus, a fundamental question is whether autism represents an aetiologically heterogeneous disorder in which the myriad genetic or environmental risk factors perturb common underlying molecular pathways in the brain. Here, we demonstrate consistent differences in transcriptome organization between autistic and normal brain by gene co-expression network analysis. Remarkably, regional patterns of gene expression that typically distinguish frontal and temporal cortex are significantly attenuated in the ASD brain, suggesting abnormalities in cortical patterning. We further identify discrete modules of co-expressed genes associated with autism: a neuronal module enriched for known autism susceptibility genes, including the neuronal specific splicing factor A2BP1 (also known as FOX1), and a module enriched for immune genes and glial markers. Using high-throughput RNA sequencing we demonstrate dysregulated splicing of A2BP1-dependent alternative exons in the ASD brain. Moreover, using a published autism genome-wide association study (GWAS) data set, we show that the neuronal module is enriched for genetically associated variants, providing independent support for the causal involvement of these genes in autism. In contrast, the immune-glial module showed no enrichment for autism GWAS signals, indicating a non-genetic aetiology for this process. Collectively, our results provide strong evidence for convergent molecular abnormalities in ASD, and implicate transcriptional and splicing dysregulation as underlying mechanisms of neuronal dysfunction in this disorder.

title: Expression Profiling of Autism Candidate Genes during Human Brain Development Implicates Central Immune Signaling Pathways
abstract: The Autism Spectrum Disorders (ASD) represent a clinically heterogeneous set of conditions with strong hereditary components. Despite substantial efforts to uncover the genetic basis of ASD, the genomic etiology appears complex and a clear understanding of the molecular mechanisms underlying Autism remains elusive. We hypothesized that focusing gene interaction networks on ASD-implicated genes that are highly expressed in the developing brain may reveal core mechanisms that are otherwise obscured by the genomic heterogeneity of the disorder. Here we report an in silico study of the gene expression profile from ASD-implicated genes in the unaffected developing human brain. By implementing a biologically relevant approach, we identified a subset of highly expressed ASD-candidate genes from which interactome networks were derived. Strikingly, immune signaling through NFκB, Tnf, and Jnk was central to ASD networks at multiple levels of our analysis, and cell-type specific expression suggested glia—in addition to neurons—deserve consideration. This work provides integrated genomic evidence that ASD-implicated genes may converge on central cytokine signaling pathways.

title: Intestinal cytokines in children with pervasive developmental disorders
abstract: A relationship between autism and gastrointestinal (GI) immune dysregulation has been postulated based on incidence of GI complaints as well as macroscopically observed lymphonodular hyperplasia and microscopically determined enterocolitis in pediatric patients with autism. To evaluate GI immunity, we quantitatively assessed levels of proinflammatory cytokines, interleukin (IL)-6, IL-8, and IL-1β, produced by intestinal biopsies of children with pervasive developmental disorders.  Fifteen patients, six with pervasive developmental disorders and nine age-matched controls, presenting for diagnostic colonoscopy were enrolled. Endoscopic biopsies were organ cultured, supernatants were harvested, and IL-6, IL-8, and IL-1β levels were quantified by ELISA. Tissue histology was evaluated by blinded pathologists.  Concentrations of IL-6 from intestinal organ culture supernatants of patients with pervasive developmental disorders (median 318.5 pg/ml, interquartile range 282.0–393.0 pg/ml) when compared with controls (median 436.9 pg/ml, interquartile range 312.6–602.5 pg/ml) were not significantly different ( p = 0.0987). Concentrations of IL-8 (median 84,000 pg/ml, interquartile range 16,000–143,000 pg/ml) when compared with controls (median 177,000 pg/ml, interquartile range 114,000–244,000 pg/ml) were not significantly different ( p = 0.0707). Concentrations of IL-1β (median 0.0 pg/ml, interquartile range 0.0–94.7 pg/ml) when compared with controls (median 0.0 pg/ml, interquartile range 0.0–60.2 pg/ml) were not significantly different ( p = 0.8826). Tissue histology was nonpathological for all patients.  We have demonstrated no significant difference in production of IL-6, IL-8, and IL-1β between patients with pervasive developmental disorders and age-matched controls. In general, intestinal levels of IL-6 and IL-8 were lower in patients with pervasive developmental disorders than in age-matched controls. These data fail to support an association between autism and GI inflammation.

title: Spontaneous Mucosal Lymphocyte Cytokine Profiles in Children with Autism and Gastrointestinal Symptoms: Mucosal Immune Activation and Reduced Counter Regulatory Interleukin-10
abstract: A lymphocytic enterocolitis has been reported in a cohort of children with autistic spectrum disorder (ASD) and gastrointestinal (GI) symptoms. This study tested the hypothesis that dysregulated intestinal mucosal immunity with enhanced pro-inflammatory cytokine production is present in these ASD children. Comparison was made with developmentally normal children with, and without, mucosal inflammation. Duodenal and colonic biopsies were obtained from 21 ASD children, and 65 developmentally normal paediatric controls, of which 38 had signs of histological inflammation. Detection of CD3+ lymphocyte staining for spontaneous intracellular TNFα, IL-2, IL-4, IFNγ, and IL-10, was performed by multicolor flow cytometry. Duodenal and colonic mucosal CD3+ lymphocyte counts were elevated in ASD children compared with noninflamed controls (p<0.03). In the duodenum, the proportion of lamina propria (LP) and epithelial CD3+TNFα+ cells in ASD children was significantly greater compared with noninflamed controls (p<0.002) but not coeliac disease controls. In addition, LP and epithelial CD3+IL-2+ and CD3+IFNγ+, and epithelial CD3+IL-4+ cells were more numerous in ASD children than in noninflamed controls (p<0.04). In contrast, CD3+IL-10+ cells were fewer in ASD children than in noninflamed controls (p<0.05). In the colon, LP CD3+TNFα+ and CD3+IFNγ+ were more frequent in ASD children than in noninflamed controls (p<0.01). In contrast with Crohn’s disease and non-Crohn’s colitis, LP and epithelial CD3+IL-10+ cells were fewer in ASD children than in nondisease controls (p<0.01). There was a significantly greater proportion of CD3+TNFα+ cells in colonic mucosa in those ASD children who had no dietary exclusion compared with those on a gluten and/or casein free diet (p<0.05). There is a consistent profile of CD3+ lymphocyte cytokines in the small and large intestinal mucosa of these ASD children, involving increased pro-inflammatory and decreased regulatory activities. The data provide further evidence of a diffuse mucosal immunopathology in some ASD children and the potential for benefit of dietary and immunomodulatory therapies.

title: Elevated cytokine levels in children with autism spectrum disorder
abstract: This study compared production of IL-2, IFN-gamma, IL-4, IL-13, IL-5 and IL-10 in peripheral blood mononuclear cells from 20 children with autism spectrum disorder to those from matched controls. Levels of all Th2 cytokines were significantly higher in cases after incubation in media alone, but the IFN-gamma/IL-13 ratio was not significantly different between cases and controls. Cases had significantly higher IL-13/IL-10 and IFN-gamma/IL-10 than controls.



Children with ASD had increased activation of both Th2 and Th1 arms of the adaptive immune response, with a Th2 predominance, and without the compensatory increase in the regulatory cytokine IL-10.

title: Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associated with impaired behavioral outcome
abstract: Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. A potential role for immune dysfunction has been suggested in ASD. To test this hypothesis, we investigated evidence of differential cytokine release in plasma samples obtained from 2 to 5 year-old children with ASD compared with age-matched typically developing (TD) children and children with developmental disabilities other than autism (DD). Participants were recruited as part of the population based case-control CHARGE (Childhood Autism Risks from Genetics and Environment) study and included: 97 participants with a confirmed diagnosis of ASD using standard assessments (DSM IV criteria and ADOS, ADI-R), 87 confirmed TD controls, and 39 confirmed DD controls. Plasma was isolated and cytokine production was assessed by multiplex Luminex™ analysis. Observations indicate significant increases in plasma levels of a number of cytokines, including IL-1β, IL-6, IL-8 and IL-12p40 in the ASD group compared with TD controls (p<0.04). Moreover, when the ASD group was separated based on the onset of symptoms, it was noted that the increased cytokine levels were predominantly in children who had a regressive form of ASD. In addition, increasing cytokine levels were associated with more impaired communication and aberrant behaviors. In conclusion, using larger number of participants than previous studies, we report significantly shifted cytokine profiles in ASD. These findings suggest that ongoing inflammatory responses may be linked to disturbances in behavior and require confirmation in larger replication studies. The characterization of immunological parameters in ASD has important implications for diagnosis, and should be considered when designing therapeutic strategies to treat core symptoms and behavioral impairments of ASD.

title: Plasma Cytokine Profiles in Subjects with High-Functioning Autism Spectrum Disorders
abstract: Accumulating evidence suggests that dysregulation of the immune system is involved in the pathophysiology of autism spectrum disorders (ASD). The aim of the study was to explore immunological markers in peripheral plasma samples from non-medicated subjects with high-functioning ASD.  A multiplex assay for cytokines and chemokines was applied to plasma samples from male subjects with high-functioning ASD (n = 28) and matched controls (n = 28). Among a total of 48 analytes examined, the plasma concentrations of IL-1β, IL-1RA, IL-5, IL-8, IL-12(p70), IL-13, IL-17 and GRO-α were significantly higher in subjects with ASD compared with the corresponding values of matched controls after correction for multiple comparisons.  The results suggest that abnormal immune responses as assessed by multiplex analysis of cytokines may serve as one of the biological trait markers for ASD.

title: Low-grade endotoxemia in patients with severe autism
abstract: The objective of this study was to examine whether levels of endotoxin and other markers of immuno-inflammatory activation are altered in adult patients with severe autism. We determined circulating serum endotoxin levels, its soluble receptor (sCD14), and markers of immuno-inflammatory activation (IL-1beta, IL-6, and IL-10) in 22 adult patients with severe autism and 28 age- and gender-matched healthy controls. Compared with healthy subjects, serum levels of endotoxin were significantly higher in autistic patients and inversely and independently correlated with Socialization scores on the Vineland Adaptive Behavior Scales (VABS) and ADI-R Domain A score (social). Whether increased endotoxin may contribute to the pathophysiology of inflammation and impaired reciprocal social interaction in autism should be further explored in future studies.

title: IL-6 is increased in the cerebellum of autistic brain and alters neural cell adhesion, migration and synaptic formation
abstract: BackgroundAlthough the cellular mechanisms responsible for the pathogenesis of autism are not understood, a growing number of studies have suggested that localized inflammation of the central nervous system (CNS) may contribute to the development of autism. Recent evidence shows that IL-6 has a crucial role in the development and plasticity of CNS.MethodsImmunohistochemistry studies were employed to detect the IL-6 expression in the cerebellum of study subjects. In vitro adenoviral gene delivery approach was used to over-express IL-6 in cultured cerebellar granule cells. Cell adhesion and migration assays, DiI labeling, TO-PRO-3 staining and immunofluorescence were used to examine cell adhesion and migration, dendritic spine morphology, cell apoptosis and synaptic protein expression respectively.ResultsIn this study, we found that IL-6 was significantly increased in the cerebellum of autistic subjects. We investigated how IL-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an IL-6 viral expression vector. We demonstrated that IL-6 over-expression in granule cells caused impairments in granule cell adhesion and migration but had little effect on the formation of dendritic spines or granule cell apoptosis. However, IL-6 over-expression stimulated the formation of granule cell excitatory synapses, without affecting inhibitory synapses.ConclusionsOur results provide further evidence that aberrant IL-6 may be associated with autism. In addition, our results suggest that the elevated IL-6 in the autistic brain could alter neural cell adhesion, migration and also cause an imbalance of excitatory and inhibitory circuits. Thus, increased IL-6 expression may be partially responsible for the pathogenesis of autism.

title: Elevation of tumor necrosis factor-alpha in cerebrospinal fluid of autistic children.
abstract: Recent reports implicating elevated cytokines in the central nervous system in a small number of patients studied with autism have reported clinical regression. These studies have not focused on tumor necrosis factor-alpha as a possible marker for inflammatory damage. A series of 10 children with autism had clinical evaluation of their serum and spinal fluid for inflammatory changes and possible metabolic disease as part of their neurological evaluation. Elevation of cerebrospinal fluid levels of tumor necrosis factor-alpha was significantly higher (mean = 104.10 pg/mL) than concurrent serum levels (mean = 2.78 pg/mL) in all of the patients studied. The ratio of the cerebrospinal fluid levels to serum levels averaged 53.7:1. This ratio is significantly higher than the elevations reported for other pathological states for which cerebrospinal fluid and serum tumor necrosis factor-alpha levels have been simultaneously measured. This observation may offer a unique insight into central nervous system inflammatory mechanisms that may contribute to the onset of autism and may serve as a potential clinical marker. More controlled study of this potentially important observation may prove valuable.

title: Safety and Observations in a Pilot Study of Lenalidomide for Treatment in Autism
abstract: Autism affects 1 : 88 children in the United States. Familial history of autoimmune disease, autoantibodies in the serum of mothers when there is more than one autistic offspring, and neuroglial response in CSF and brain tissue in autistic patients suggest an immunological variable may be associated with this condition. Lenalidomide has the potential to invoke changes in TNF-α with less toxicity than thalidomide. This pilot study evaluated lenalidomide at reduction of TNF-α and improvement of behavior and language in children with autism with elevated TNF-α. Subjects with elevated TNF-α were given 2.5 mgs lenalidomide daily for 12-weeks. Pharmacodynamics and safety was evaluated. Changes in language and autistic behaviors after six and twelve weeks were measured. Although statistical significance was not achieved for most measures, there were trends toward improvement. After 6-weeks, mean receptive language increased: 60.67 ± 12.06 to 65.00 ± 15.10 (P = 0.11) and symptoms of autism decreased (40.75 ± 5.96 versus 38.67 ± 7.90, P = 0.068). After 12-weeks, CSF-TNF-α declined 57% ± 25% from 80.5 ± 41.03 to 38.0 ± 31.27 (P = 0.068). Serum TNF-α declined 57% (92.50 ± 68.92 to 40.25 ± 44.53 (P = 0.048). This study suggests that lenalidomide is tolerated as a treatment by children with autism and should be further studied as a potential agent for cytockine inflammation.

title: Brief Report: Autistic Disorder in Three Children with Cytomegalovirus Infection
abstract: Previous research has identified a relationship between autistic disorder (autism) and specific congenital infections. Three cases of congenital or perinatal cytomegalovirus (CMV) infection occurring in association with autism are described. Hypothetical mechanisms relating congenital infection, such as CMV, to the development of autism are discussed. A better understanding of the immunologic response to certain congenital infections may provide important information pertaining to the pathophysiology and etiology of autism in vulnerable individuals.

title: Plasma increase of interleukin-12 and interferon-gamma. Pathological significance in autism
abstract: Immune factors such as autoimmunity have been implicated in the genesis of autism, a neurodevelopmental disorder. Since autoimmune response involves immune activation, the plasma levels of interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), interleukin-12 (IL-12), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and soluble intercellular adhesion molecule-1 (sICAM-1) were measured in autistic patients and age-matched normal controls. The levels of IL-12 and IFN-gamma were significantly (P < or = 0.05) higher in patients as compared to controls. However, IFN-alpha, IL-6, TNF-alpha, and sICAM-1 levels did not significantly differ between the two groups. Because macrophage-derived IL-12 is known to selectively induce IFN-gamma in T helper type-1 (Th-1) cells, it is suggested that IL-12 and IFN-gamma increases may indicate antigenic stimulation of Th-1 cells pathogenetically linked to autoimmunity in autism.

title: Metabolic biomarkers related to oxidative stress and antioxidant status in Saudi autistic children.
abstract: Measurement of oxidative stress and antioxidant-related parameters (enzymatic and non-enzymatic) in Saudi autistic children.



30 autistic children (22 males and 8 females) aged 3-15 years (25/30 of these were below 8 years old), and 30 healthy children as control group were included in this study. Levels of lipid peroxides, vitamin E, vitamin C, glutathione together with enzymatic activities of glutathione peroxidase (GSH-Px), and catalase were determined in plasma while superoxide dismutase (SOD was measured in red blood cells of both groups.



Lipid peroxidation was found to be significantly higher in autistic compared to control Saudi children. On the other hand, vitamin E and glutathione were remarkably lower in autistic patients while vitamin C shows non-significant lower values. Regarding the enzymatic antioxidants, both glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were significantly higher in autistic compared to control while catalase recorded more or less similar activities in both groups.



Saudi autistic children are under H(2)O(2) stress due to GSH depletion, over expression of SOD together with the unchanged catalase enzyme. This could be helpful in the early diagnosis of young autistic patients and suggesting the possibility of antioxidant supplementation for the early intervention with autistic children.

title: Metabolic Imbalance Associated with Methylation Dysregulation and Oxidative Damage in Children with Autism
abstract: Oxidative stress and abnormal DNA methylation have been implicated in the pathophysiology of autism. We investigated the dynamics of an integrated metabolic pathway essential for cellular antioxidant and methylation capacity in 68 children with autism, 54 age-matched control children and 40 unaffected siblings. The metabolic profile of unaffected siblings differed significantly from case siblings but not from controls. Oxidative protein/DNA damage and DNA hypomethylation (epigenetic alteration) were found in autistic children but not paired siblings or controls. These data indicate that the deficit in antioxidant and methylation capacity is specific for autism and may promote cellular damage and altered epigenetic gene expression. Further, these results suggest a plausible mechanism by which pro-oxidant environmental stressors may modulate genetic predisposition to autism.

title: Intracellular and Extracellular Redox Status and Free Radical Generation in Primary Immune Cells from Children with Autism
abstract: The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation. To investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children. These data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children. These results suggest that the loss of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism.

title: A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures
abstract: Recent studies have implicated physiological and metabolic abnormalities in autism spectrum disorders (ASD) and other psychiatric disorders, particularly immune dysregulation or inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures (‘four major areas’). The aim of this study was to determine trends in the literature on these topics with respect to ASD. A comprehensive literature search from 1971 to 2010 was performed in these four major areas in ASD with three objectives. First, publications were divided by several criteria, including whether or not they implicated an association between the physiological abnormality and ASD. A large percentage of publications implicated an association between ASD and immune dysregulation/inflammation (416 out of 437 publications, 95%), oxidative stress (all 115), mitochondrial dysfunction (145 of 153, 95%) and toxicant exposures (170 of 190, 89%). Second, the strength of evidence for publications in each area was computed using a validated scale. The strongest evidence was for immune dysregulation/inflammation and oxidative stress, followed by toxicant exposures and mitochondrial dysfunction. In all areas, at least 45% of the publications were rated as providing strong evidence for an association between the physiological abnormalities and ASD. Third, the time trends in the four major areas were compared with trends in neuroimaging, neuropathology, theory of mind and genetics (‘four comparison areas’). The number of publications per 5-year block in all eight areas was calculated in order to identify significant changes in trends. Prior to 1986, only 12 publications were identified in the four major areas and 51 in the four comparison areas (42 for genetics). For each 5-year period, the total number of publications in the eight combined areas increased progressively. Most publications (552 of 895, 62%) in the four major areas were published in the last 5 years (2006–2010). Evaluation of trends between the four major areas and the four comparison areas demonstrated that the largest relative growth was in immune dysregulation/inflammation, oxidative stress, toxicant exposures, genetics and neuroimaging. Research on mitochondrial dysfunction started growing in the last 5 years. Theory of mind and neuropathology research has declined in recent years. Although most publications implicated an association between the four major areas and ASD, publication bias may have led to an overestimation of this association. Further research into these physiological areas may provide insight into general or subset-specific processes that could contribute to the development of ASD and other psychiatric disorders.

title: Abnormal Transmethylation/transsulfuration Metabolism and DNA Hypomethylation Among Parents of Children with Autism
abstract: An integrated metabolic profile reflects the combined influence of genetic, epigenetic, and environmental factors that affect the candidate pathway of interest. Recent evidence suggests that some autistic children may have reduced detoxification capacity and may be under chronic oxidative stress. Based on reports of abnormal methionine and glutathione metabolism in autistic children, it was of interest to examine the same metabolic profile in the parents. The results indicated that parents share similar metabolic deficits in methylation capacity and glutathione-dependent antioxidant/detoxification capacity observed in many autistic children. Studies are underway to determine whether the abnormal profile in parents reflects linked genetic polymorphisms in these pathways or whether it simply reflects the chronic stress of coping with an autistic child.

title: Glutathione pathway gene variation and risk of autism spectrum disorders
abstract: Despite evidence that autism is highly heritable with estimates of 15 or more genes involved, few studies have directly examined associations of multiple gene interactions. Since inability to effectively combat oxidative stress has been suggested as a mechanism of autism, we examined genetic variation 42 genes (308 single-nucleotide polymorphisms (SNPs)) related to glutathione, the most important antioxidant in the brain, for both marginal association and multi-gene interaction among 318 case–parent trios from The Autism Genetic Resource Exchange. Models of multi-SNP interactions were estimated using the trio Logic Regression method. A three-SNP joint effect was observed for genotype combinations of SNPs in glutaredoxin, glutaredoxin 3 (GLRX3), and cystathione gamma lyase (CTH); OR = 3.78, 95% CI: 2.36, 6.04. Marginal associations were observed for four genes including two involved in the three-way interaction: CTH, alcohol dehydrogenase 5, gamma-glutamylcysteine synthetase, catalytic subunit and GLRX3. These results suggest that variation in genes involved in counterbalancing oxidative stress may contribute to autism, though replication is necessary.

title: Oxidative stress-related biomarkers in autism: systematic review and meta-analyses.
abstract: Autism spectrum disorders (ASDs) are rarely diagnosed in children younger than 2 years, because diagnosis is based entirely on behavioral tests. Oxidative damage may play a central role in this pathogenesis, together with the interconnected transmethylation cycle and transsulfuration pathway. In an attempt to clarify and quantify the relationship between oxidative stress-related blood biomarkers and ASDs, a systematic literature review was carried out. For each identified study, mean biomarker levels were compared in cases and controls providing a point estimate, the mean ratio, for each biomarker. After meta-analysis, the ASD patients showed decreased blood levels of reduced glutathione (27%), glutathione peroxidase (18%), methionine (13%), and cysteine (14%) and increased concentrations of oxidized glutathione (45%) relative to controls, whereas superoxide dismutase, homocysteine, and cystathionine showed no association with ASDs. For the C677T allele in the methylene tetrahydrofolate reductase gene (MTHFR), homozygous mutant subjects (TT) showed a meta-OR of 2.26 (95% CI 1.30-3.91) of being affected by ASD with respect to the homozygous nonmutant (CC). Case-control studies on blood levels of vitamins suggest a lack of association (folic acid and vitamin B12) or rare association (vitamins A, B6, C, D, E). Sparse results were available for other biomarkers (ceruloplasmin, catalase, cysteinylglycine, thiobarbituric acid-reactive substances, nitric oxide) and for polymorphisms in other genes. Existing evidence is heterogeneous and many studies are limited by small sample size and effects. In conclusion, existing evidence suggests a role for glutathione metabolism, the transmethylation cycle, and the transsulfuration pathway, although these findings should be interpreted with caution, and larger, more standardized studies are warranted.

title: The MTHFR 677C→T polymorphism and behaviors in children with autism: exploratory genotype–phenotype correlations
abstract: New evidence suggests that autism may be associated with (a) varied behavioral responses to folate therapy and (b) metabolic anomalies, including those in folate metabolism, that contribute to hypomethylation of DNA. We hypothesized that children with autism who are homozygous for the MTHFR 677 T allele (TT) and, to a lesser extent those with the CT variant, would exhibit more behavioral problems and/or more severe problematic behaviors than homozygous wild‐type (CC) individuals because of difficulties in effectively converting 5,10‐MTHF to 5‐MTHF. Data from the Autism Genetic Resource Exchange (AGRE) collection were analyzed for all children who met strict criteria for autism per the Autism Diagnostic Interview—Revised (ADI‐R) and the Autism Diagnostic Observation Schedule (ADOS) and who had been genotyped for the 677 C to T MTHFR polymorphism (n=147). Chi‐square tests, logistic regression, and one‐way ANOVAs were used to determine whether differences existed among MTHFR genotypes for specific behaviors on the ADI‐R and indices for level of functioning. Exploratory results indicated four behaviors from the ADI‐R that were more common and problematic (95% CI) among those with at least one copy of the T allele as compared to homozygous wild‐type individuals: direct gaze, current complex body movements, a history of self‐injurious behavior, and current overactivity (ORs=2.72, 2.33, 2.12, 2.47, respectively). No differences existed among genotypes for level of functioning as measured with the Peabody Picture Vocabulary Test—Third Edition, Ravens Colored Progressive Matrices, or the Vineland Adaptive Behavior Scales. Findings call for further investigation of the relationship between folate metabolism and problem behaviors among children with autism.

title: Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism.
abstract: Autism is a complex neurodevelopmental disorder that usually presents in early childhood and that is thought to be influenced by genetic and environmental factors. Although abnormal metabolism of methionine and homocysteine has been associated with other neurologic diseases, these pathways have not been evaluated in persons with autism.



The purpose of this study was to evaluate plasma concentrations of metabolites in the methionine transmethylation and transsulfuration pathways in children diagnosed with autism.



Plasma concentrations of methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), adenosine, homocysteine, cystathionine, cysteine, and oxidized and reduced glutathione were measured in 20 children with autism and in 33 control children. On the basis of the abnormal metabolic profile, a targeted nutritional intervention trial with folinic acid, betaine, and methylcobalamin was initiated in a subset of the autistic children.



Relative to the control children, the children with autism had significantly lower baseline plasma concentrations of methionine, SAM, homocysteine, cystathionine, cysteine, and total glutathione and significantly higher concentrations of SAH, adenosine, and oxidized glutathione. This metabolic profile is consistent with impaired capacity for methylation (significantly lower ratio of SAM to SAH) and increased oxidative stress (significantly lower redox ratio of reduced glutathione to oxidized glutathione) in children with autism. The intervention trial was effective in normalizing the metabolic imbalance in the autistic children.



An increased vulnerability to oxidative stress and a decreased capacity for methylation may contribute to the development and clinical manifestation of autism.

title: Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism.
abstract: Metabolic abnormalities and targeted treatment trials have been reported for several neurobehavioral disorders but are relatively understudied in autism.



The objective of this study was to determine whether or not treatment with the metabolic precursors, methylcobalamin and folinic acid, would improve plasma concentrations of transmethylation/transsulfuration metabolites and glutathione redox status in autistic children.



In an open-label trial, 40 autistic children were treated with 75 microg/kg methylcobalamin (2 times/wk) and 400 microg folinic acid (2 times/d) for 3 mo. Metabolites in the transmethylation/transsulfuration pathway were measured before and after treatment and compared with values measured in age-matched control children.



The results indicated that pretreatment metabolite concentrations in autistic children were significantly different from values in the control children. The 3-mo intervention resulted in significant increases in cysteine, cysteinylglycine, and glutathione concentrations (P < 0.001). The oxidized disulfide form of glutathione was decreased and the glutathione redox ratio increased after treatment (P < 0.008). Although mean metabolite concentrations were improved significantly after intervention, they remained below those in unaffected control children.



The significant improvements observed in transmethylation metabolites and glutathione redox status after treatment suggest that targeted nutritional intervention with methylcobalamin and folinic acid may be of clinical benefit in some children who have autism.

title: Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features
abstract: It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na+/K+-ATPase activity (−66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.

title: The Severity of Autism Is Associated with Toxic Metal Body Burden and Red Blood Cell Glutathione Levels
abstract: This study investigated the relationship of children's autism symptoms with their toxic metal body burden and red blood cell (RBC) glutathione levels. In children ages 3–8 years, the severity of autism was assessed using four tools: ADOS, PDD-BI, ATEC, and SAS. Toxic metal body burden was assessed by measuring urinary excretion of toxic metals, both before and after oral dimercaptosuccinic acid (DMSA). Multiple positive correlations were found between the severity of autism and the urinary excretion of toxic metals. Variations in the severity of autism measurements could be explained, in part, by regression analyses of urinary excretion of toxic metals before and after DMSA and the level of RBC glutathione (adjusted R 2 of 0.22–0.45, P < .005 in all cases). This study demonstrates a significant positive association between the severity of autism and the relative body burden of toxic metals.

title: Enrichment of Elevated Plasma F2t-Isoprostane Levels in Individuals with Autism Who Are Stratified by Presence of Gastrointestinal Dysfunction
abstract: Etiology is unknown in the majority of individuals with autism spectrum disorder (ASD). One strategy to investigate pathogenesis is to stratify this heterogeneous disorder based on a prominent phenotypic feature that enriches for homogeneity within population strata. Co-occurring gastrointestinal dysfunction (GID) characterizes a subset of children with ASD. Our current objective was to investigate a potential pathophysiological measure to test the hypothesis that children with both ASD and GID have a more severe metabolic dysfunction than children with ASD-only, given that the highly metabolically active brain and gastrointestinal system may additively contribute measurable impairment. Plasma levels of F2t-Isoprostanes (F2-IsoPs), a gold standard biomarker of oxidative stress, were measured in 87 children in four groups: ASD-GID, ASD-only, GID-only and Unaffected. F2-IsoP levels were elevated in all 3 clinical groups compared to the Unaffected group, with the ASD-GID group significantly elevated above the ASD-only group (mean, SD in pg/mg: ASD-GID 53.6, 24.4; ASD-only 36.5, 13.3; p = 0.007). Adjusting for age, sex, and triglyceride levels, F2-IsoP levels remained significantly different between study groups, with a moderate effect size of ηp 2 = 0.187 (p = 0.001). Elevation in peripheral oxidative stress is consistent with, and may contribute to, the more severe functional impairments in the ASD-GID group. With unique medical, metabolic, and behavioral features in children with ASD-GID, the present findings serve as a compelling rationale for both individualized approaches to clinical care and integrated studies of biomarker enrichment in ASD subgroups that may better address the complex etiology of ASD.

title: Brain region‐specific deficit in mitochondrial electron transport chain complexes in children with autism
abstract: (2011) 117, 209–220.

title: Brain Region-Specific Changes in Oxidative Stress and Neurotrophin Levels in Autism Spectrum Disorders (ASD)
abstract: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by social and language deficits, stereotypic behavior, and abnormalities in motor functions. The particular set of behavioral impairments expressed in any given individual is variable across the spectrum. These behavioral abnormalities are consistent with our current understanding of the neuropathology of ASD which suggests abnormalities in the amygdala, temporal and frontal cortexes, hippocampus, and cerebellum. However, regions unrelated to these behavioral deficits appear largely intact. Both genetic predisposition and environmental toxins and toxicants have been implicated in the etiology of autism; the impact of these environmental triggers is associated with increases in oxidative stress, and is further exacerbated when combined with genetic susceptibility. We have previously reported increased levels of 3-nitrotyrosine (3-NT), a marker of oxidative stress, in ASD cerebella. We have also shown that this increase was associated with an elevation in neurotrophin-3 (NT-3) levels. The objectives of the current study were to determine whether the increase in oxidative stress in ASD is brain region-specific, to identify the specific brain regions affected by oxidative stress, and to compare brain region-specific NT-3 expression between ASD and control cases. The levels of 3-NT and NT-3 were measured with specific ELISAs in individual brain regions of two autistic and age- and postmortem interval (PMI)--matched control donors. In the control brain, the levels of 3-NT were uniformly low in all brain regions examined ranging from 1.6 to 12.0 pmol/g. On the other hand, there was a great variation in 3-NT levels between individual brain regions of the autistic brains ranging from 1.7 to 281.2 pmol/g. The particular brain regions with the increased 3-NT and the magnitude of the increase were both different in the two autistic cases. In the older autistic case, the brain regions with highest levels of 3-NT included the orbitofrontal cortex (214.5 pmol/g), Wernicke’s area (171.7 pmol/g), cerebellar vermis (81.2 pmol/g), cerebellar hemisphere (37.2 pmol/g), and pons (13.6 pmol/g); these brain areas are associated with the speech processing, sensory and motor coordination, emotional and social behavior, and memory. Brain regions that showed 3-NT increase in both autistic cases included the cerebellar hemispheres and putamen. Consistent with our earlier report, we found an increase in NT-3 levels in the cerebellar hemisphere in both autistic cases. We also detected an increase in NT-3 level in the dorsolateral prefrontal cortex (BA46) in the older autistic case and in the Wernicke’s area and cingulate gyrus in the younger case. These preliminary results reveal, for the first time, brain region-specific changes in oxidative stress marker 3-NT and neurotrophin-3 levels in ASD.

title: Brain Region-Specific Glutathione Redox Imbalance in Autism
abstract: Autism is a heterogeneous, behaviorally defined neurodevelopmental disorder. Recently, we reported a brain region–specific increase in lipid peroxidation, and deficits in mitochondrial electron transport chain complexes in autism, suggesting the role of oxidative stress and mitochondrial dysfunction in the pathophysiology of autism. However, the antioxidant status of the brain is not known in autism. Glutathione is a major endogenous antioxidant that plays a crucial role in protecting cells from exogenous and endogenous toxins, particularly in the central nervous system. The present study examines the concentrations of glutathione (GSH, reduced form; and GSSG, oxidized form) and the redox ratio of GSH to GSSG (marker of oxidative stress) in different regions of brains from autistic subjects and age-matched control subjects. In the cerebellum and temporal cortex from subjects with autism, GSH levels were significantly decreased by 34.2 and 44.6 %, with a concomitant increase in the levels of GSSG by 38.2 and 45.5 %, respectively, as compared to the control group. There was also a significant decrease in the levels of total GSH (tGSH) by 32.9 % in the cerebellum, and by 43.1 % in the temporal cortex of subjects with autism. In contrast, there was no significant change in GSH, GSSG and tGSH levels in the frontal, parietal and occipital cortices in autism versus control group. The redox ratio of GSH to GSSG was also significantly decreased by 52.8 % in the cerebellum and by 60.8 % in the temporal cortex of subjects with autism, suggesting glutathione redox imbalance in the brain of individuals with autism. These findings indicate that autism is associated with deficits in glutathione antioxidant defense in selective regions of the brain. We suggest that disturbances in brain glutathione homeostasis may contribute to oxidative stress, immune dysfunction and apoptosis, particularly in the cerebellum and temporal lobe, and may lead to neurodevelopmental abnormalities in autism.

title: Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain
abstract: Despite increasing evidence of oxidative stress in the pathophysiology of autism, most studies have not evaluated biomarkers within specific brain regions, and the functional consequences of oxidative stress remain relatively understudied. We examined frozen samples from the cerebellum and temporal cortex (Brodmann area 22 (BA22)) from individuals with autism and unaffected controls (n=15 and n=12 per group, respectively). Biomarkers of oxidative stress, including reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione redox/antioxidant capacity (GSH/GSSG), were measured. Biomarkers of oxidative protein damage (3-nitrotyrosine; 3-NT) and oxidative DNA damage (8-oxo-deoxyguanosine; 8-oxo-dG) were also assessed. Functional indicators of oxidative stress included relative levels of 3-chlorotyrosine (3-CT), an established biomarker of a chronic inflammatory response, and aconitase activity, a biomarker of mitochondrial superoxide production. Consistent with previous studies on plasma and immune cells, GSH and GSH/GSSG were significantly decreased in both autism cerebellum (P<0.01) and BA22 (P<0.01). There was a significant increase in 3-NT in the autism cerebellum and BA22 (P<0.01). Similarly, 8-oxo-dG was significantly increased in autism cerebellum and BA22 (P<0.01 and P=0.01, respectively), and was inversely correlated with GSH/GSSG in the cerebellum (P<0.01). There was a significant increase in 3-CT levels in both brain regions (P<0.01), whereas aconitase activity was significantly decreased in autism cerebellum (P<0.01), and was negatively correlated with GSH/GSSG (P=0.01). Together, these results indicate that decreased GSH/GSSG redox/antioxidant capacity and increased oxidative stress in the autism brain may have functional consequence in terms of a chronic inflammatory response, increased mitochondrial superoxide production, and oxidative protein and DNA damage.

title: Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism
abstract: Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence suggests that mitochondrial dysfunction and oxidative stress may contribute to the etiology of autism. This is the first study to compare the activities of mitochondrial electron transport chain (ETC) complexes (I–V) and pyruvate dehydrogenase (PDH), as well as mitochondrial DNA (mtDNA) copy number in the frontal cortex tissues from autistic and age-matched control subjects. The activities of complexes I, V and PDH were most affected in autism (n=14) being significantly reduced by 31%, 36% and 35%, respectively. When 99% confidence interval (CI) of control group was taken as a reference range, impaired activities of complexes I, III and V were observed in 43%, 29% and 43% of autistic subjects, respectively. Reduced activities of all five ETC complexes were observed in 14% of autistic cases, and the activities of multiple complexes were decreased in 29% of autistic subjects. These results suggest that defects in complexes I and III (sites of mitochondrial free radical generation) and complex V (adenosine triphosphate synthase) are more prevalent in autism. PDH activity was also reduced in 57% of autistic subjects. The ratios of mtDNA of three mitochondrial genes ND1, ND4 and Cyt B (that encode for subunits of complexes I and III) to nuclear DNA were significantly increased in autism, suggesting a higher mtDNA copy number in autism. Compared with the 95% CI of the control group, 44% of autistic children showed higher copy numbers of all three mitochondrial genes examined. Furthermore, ND4 and Cyt B deletions were observed in 44% and 33% of autistic children, respectively. This study indicates that autism is associated with mitochondrial dysfunction in the brain.

title: Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.
abstract: Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM.

title: Mitochondrial abnormalities in temporal lobe of autistic brain
abstract: Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear.

title: Mitochondrial dysfunction in autism.
abstract: Impaired mitochondrial function may influence processes highly dependent on energy, such as neurodevelopment, and contribute to autism. No studies have evaluated mitochondrial dysfunction and mitochondrial DNA (mtDNA) abnormalities in a well-defined population of children with autism.



To evaluate mitochondrial defects in children with autism.



Observational study using data collected from patients aged 2 to 5 years who were a subset of children participating in the Childhood Autism Risk From Genes and Environment study in California, which is a population-based, case-control investigation with confirmed autism cases and age-matched, genetically unrelated, typically developing controls, that was launched in 2003 and is still ongoing. Mitochondrial dysfunction and mtDNA abnormalities were evaluated in lymphocytes from 10 children with autism and 10 controls.



Oxidative phosphorylation capacity, mtDNA copy number and deletions, mitochondrial rate of hydrogen peroxide production, and plasma lactate and pyruvate.



The reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (normalized to citrate synthase activity) in lymphocytic mitochondria from children with autism was significantly lower compared with controls (mean, 4.4 [95% confidence interval {CI}, 2.8-6.0] vs 12 [95% CI, 8-16], respectively; P = .001). The majority of children with autism (6 of 10) had complex I activity below control range values. Higher plasma pyruvate levels were found in children with autism compared with controls (0.23 mM [95% CI, 0.15-0.31 mM] vs 0.08 mM [95% CI, 0.04-0.12 mM], respectively; P = .02). Eight of 10 cases had higher pyruvate levels but only 2 cases had higher lactate levels compared with controls. These results were consistent with the lower pyruvate dehydrogenase activity observed in children with autism compared with controls (1.0 [95% CI, 0.6-1.4] nmol × [min × mg protein](-1) vs 2.3 [95% CI, 1.7-2.9] nmol × [min × mg protein](-1), respectively; P = .01). Children with autism had higher mitochondrial rates of hydrogen peroxide production compared with controls (0.34 [95% CI, 0.26-0.42] nmol × [min × mg of protein](-1) vs 0.16 [95% CI, 0.12-0.20] nmol × [min × mg protein](-1) by complex III; P = .02). Mitochondrial DNA overreplication was found in 5 cases (mean ratio of mtDNA to nuclear DNA: 239 [95% CI, 217-239] vs 179 [95% CI, 165-193] in controls; P = 10(-4)). Deletions at the segment of cytochrome b were observed in 2 cases (ratio of cytochrome b to ND1: 0.80 [95% CI, 0.68-0.92] vs 0.99 [95% CI, 0.93-1.05] for controls; P = .01).



In this exploratory study, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children.

title: Fever Plus Mitochondrial Disease Could Be Risk Factors for Autistic Regression
abstract: Autistic spectrum disorders encompass etiologically heterogeneous persons, with many genetic causes. A subgroup of these individuals has mitochondrial disease. Because a variety of metabolic disorders, including mitochondrial disease show regression with fever, a retrospective chart review was performed and identified 28 patients who met diagnostic criteria for autistic spectrum disorders and mitochondrial disease. Autistic regression occurred in 60.7% (17 of 28), a statistically significant increase over the general autistic spectrum disorder population (P < .0001). Of the 17 individuals with autistic regression, 70.6% (12 of 17) regressed with fever and 29.4% (5 of 17) regressed without identifiable linkage to fever or vaccinations. None showed regression with vaccination unless a febrile response was present. Although the study is small, a subgroup of patients with mitochondrial disease may be at risk of autistic regression with fever. Although recommended vaccinations schedules are appropriate in mitochondrial disease, fever management appears important for decreasing regression risk.

title: Mitochondrial DNA and anti-mitochondrial antibodies in serum of autistic children
abstract: Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by difficulties in communication, cognitive and learning deficits, as well as stereotypic behaviors. For the majority of cases there are no reliable biomarkers or distinct pathogenesis. However, increasing evidence indicates ASD may be associated with some immune dysregulation, and may have a neuroimmune component. We recently showed that the peptide neurotensin (NT) is increased in autistic children. We now show that NT induces release of extracellular mitochondrial DNA (mtDNA) that could act as "autoimmune" trigger. We further show that serum from young autistic patients contains mtDNA (n = 20; cytochrome B, p = 0.0002 and 7S, p = 0.006), and anti-mitochondrial antibody Type 2 (n = 14; p = 0.001) as compared to normally developing, unrelated controls (n = 12). Extracellular blood mtDNA and other components may characterize an autistic endophenotype and may contribute to its pathogenesis by activating autoimmune responses.

title: Genetics and Mitochondrial Abnormalities in Autism Spectrum Disorders: A Review
abstract: We review the current status of the role and function of the mitochondrial DNA (mtDNA) in the etiology of autism spectrum disorders (ASD) and the interaction of nuclear and mitochondrial genes. High lactate levels reported in about one in five children with ASD may indicate involvement of the mitochondria in energy metabolism and brain development. Mitochondrial disturbances include depletion, decreased quantity or mutations of mtDNA producing defects in biochemical reactions within the mitochondria. A subset of individuals with ASD manifests copy number variation or small DNA deletions/duplications, but fewer than 20 percent are diagnosed with a single gene condition such as fragile X syndrome. The remaining individuals with ASD have chromosomal abnormalities (e.g., 15q11-q13 duplications), other genetic or multigenic causes or epigenetic defects. Next generation DNA sequencing techniques will enable better characterization of genetic and molecular anomalies in ASD, including defects in the mitochondrial genome particularly in younger children.

title: Redox metabolism abnormalities in autistic children associated with mitochondrial disease
abstract: Research studies have uncovered several metabolic abnormalities associated with autism spectrum disorder (ASD), including mitochondrial disease (MD) and abnormal redox metabolism. Despite the close connection between mitochondrial dysfunction and oxidative stress, the relation between MD and oxidative stress in children with ASD has not been studied. Plasma markers of oxidative stress and measures of cognitive and language development and ASD behavior were obtained from 18 children diagnosed with ASD who met criteria for probable or definite MD per the Morava et al. criteria (ASD/MD) and 18 age and gender-matched ASD children without any biological markers or symptoms of MD (ASD/NoMD). Plasma measures of redox metabolism included reduced free glutathione (fGSH), oxidized glutathione (GSSG), the fGSH/GSSG ratio and 3-nitrotyrosine (3NT). In addition, a plasma measure of chronic immune activation, 3-chlorotyrosine (3CT), was also measured. Language was measured using the preschool language scale or the expressive one-word vocabulary test (depending on the age), adaptive behaviour was measured using the Vineland Adaptive Behavior Scale (VABS) and core autism symptoms were measured using the Autism Symptoms Questionnaire and the Social Responsiveness Scale. Children with ASD/MD were found to have lower scores on the communication and daily living skill subscales of the VABS despite having similar language and ASD symptoms. Children with ASD/MD demonstrated significantly higher levels of fGSH/GSSG and lower levels of GSSG as compared with children with ASD/NoMD, suggesting an overall more favourable glutathione redox status in the ASD/MD group. However, compare with controls, both ASD groups demonstrated lower fGSH and fGSH/GSSG, demonstrating that both groups suffer from redox abnormalities. Younger ASD/MD children had higher levels of 3CT than younger ASD/NoMD children because of an age-related effect in the ASD/MD group. Both ASD groups demonstrated significantly higher 3CT levels than control subjects, suggesting that chronic inflammation was present in both groups of children with ASD. Interestingly, 3NT was found to correlate positively with several measures of cognitive function, development and behavior for the ASD/MD group, but not the ASD/NoMD group, such that higher 3NT concentrations were associated with more favourable adaptive behaviour, language and ASD-related behavior. To determine whether difference in receiving medications and/or supplements could account for the differences in redox and inflammatory biomarkers across ASD groups, we examined differences in medication and supplements across groups and their effect of redox and inflammatory biomarkers. Overall, significantly more participants in the ASD/MD group were receiving folate, vitamin B12, carnitine, co-enzyme Q10, B vitamins and antioxidants. We then determined whether folate, carnitine, co-enzyme Q10, B vitamins and/or antioxidants influenced redox or inflammatory biomarkers. Antioxidant supplementation was associated with a significantly lower GSSG, whereas antioxidants, co-enzyme Q10 and B vitamins were associated with a higher fGSH/GSSG ratio. There was no relation between folate, carnitine, co-enzyme Q10, B vitamins and antioxidants with 3NT, 3CT or fGSH. Overall, our findings suggest that ASD/MD children with a more chronic oxidized microenvironment have better development. We interpret this finding in light of the fact that more active mitochondrial can create a greater oxidized microenvironment especially when dysfunctional. Thus, compensatory upregulation of mitochondria which are dysfunctional may both increase activity and function at the expense of a more oxidized microenvironment. Although more ASD/MD children were receiving certain supplements, the use of such supplements were not found to be related to the redox biomarkers that were related to cognitive development or behavior in the ASD/MD group but could possibly account for the difference in glutathione metabolism noted between groups. This study suggests that different subgroups of children with ASD have different redox abnormalities, which may arise from different sources. A better understanding of the relationship between mitochondrial dysfunction in ASD and oxidative stress, along with other factors that may contribute to oxidative stress, will be critical to understanding how to guide treatment and management of ASD children. This study also suggests that it is important to identify ASD/MD children as they may respond differently to specific treatments because of their specific metabolic profile.

title: Mitochondrial physiology and autism spectrum disorder
abstract: Recent studies have suggested that mitochondrial dysfunction is relatively common in many children with autism spectrum disorder (ASD) and appears to be the most prevalent metabolic disorder associated with ASD. However, the exact prevalence of mitochondrial disease in ASD is not clear as the classic criteria for diagnosing mitochondrial disease in ASD appear to underestimate the true prevalence. Indeed, the prevalence of biomarkers of mitochondrial dysfunction and abnormal electron transport chain function appears to be high in ASD. In addition, recent studies have uncovered novel forms of mitochondrial dysfunction in ASD that may not be readily recognized by classic diagnostic criteria. This critical review provides a brief overview of mitochondrial function and its influence on other cellular systems in the context of ASD. The mitochondria's role in producing energy is complex and linked to other metabolic systems. Mitochondrial energy production is a result of the tricarboxylic acid cycle, fatty-acid oxidation and the electron transport chain working in concert. The unique architecture of the mitochondria facilitates the function of these systems. The function and architecture of the mitochondria for producing energy with a special reference to the source of biomarkers of mitochon-drial dysfunction is reviewed. Non-energy functions of the mitochon-dria including calcium buffering,

title: A preliminary 31P MRS study of autism: Evidence for undersynthesis and increased degradation of brain membranes
abstract: In this pilot study, brain high energy phosphate and membrane phospholipid metabolism were investigated in the dorsal prefrontal cortex of 11 high-functioning autistic adolescent and young adult men (the age range is 12-36 years) and 11 age-, gender-, IQ, race- and socioeconomic status-matched normal controls using in vivo 31P nuclear magnetic resonance spectroscopy (MRS). The autistic group had decreased levels of phosphocreatine and esterified ends (alpha ATP + alpha ADP + dinucleotides + diphosphosugars) compared to the controls. When the metabolite levels were compared within each subject group with neuropsychologic and language test scores, a common pattern of correlations was observed across measures in the autistic group, but not in the control group. As test performance declined in the autistic subjects, levels of the most labile high energy phosphate compound and of membrane building blocks decreased, and levels of membrane breakdown products increased. No significant correlations were present with age in either group or with IQ in the control group, suggesting that these findings were not the consequence of age or IQ effects. This pilot study provides tentative evidence of alterations in brain energy and phospholipid metabolism in autism that correlate with the neuropsychologic and language deficits. The findings are consistent with a hypermetabolic energy state and undersynthesis of brain membranes and may relate to the neurophysiologic and neuropathologic abnormalities in autism.

title: Biomarkers of Abnormal Energy Metabolism in Children with Autism Spectrum Disorder
abstract: Biomarkers of mitochondrial disease were studies in 133 consecutive autism spectrum disorder patients to determine the prevalence of abnormalities in biomarkers of mitochondrial disease. Biomarkers included traditional biomarkers of mitochondrial disease (lactate, alanine), fatty-acid oxidation defects (acyl-carnitine panel) and recently described novel biomarkers of detecting mitochondrial dysfunction in individuals with autism spectrum disorder (alanine-to-lysine ratio, creatine kinase, aspartate transaminase). Biomarkers were collected in the morning fasting state. Abnormal biomarker values were verified by repeat testing. For those with abnormal acyl-carnitine panels, secondary disorders of fatty acid metabolism were ruled out. Abnormalities in lactate, alanine-to-lysine ratio and acyl-carnitine panels occurred in over 30% of children on initial testing. Among the patients with abnormal biomarkers who had repeated testing, abnormalities were confirmed about half of the time except for alanine which was only confirmed 20% of the time. Elevation in alanine-to-lysine ratio was associated with epilepsy and elevation in multiple acyl-carnitines was associated with regression. In order to confirm the significance of certain biomarkers, a wide variety of mitochondrial biomarker values were compared between specific subgroups of children with abnormal biomarkers and matched children without any abnormalities in biomarkers. Lactate, alanine-to-lysine ratio and acyl-carnitine panel groups demonstrated abnormalities in multiple mitochondrial biomarkers, confirming the validity of these biomarkers of mitochondrial dysfunction. This study demonstrates that multiple biomarkers of mitochondrial dysfunction are elevated in a significant portion of children with autism spectrum disorder and lend support to the notion that disorders of energy production may affect a significant subset of children with autism.

title: Altered calcium homeostasis in autism-spectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1
abstract: Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient–control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium (Ca2+). AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca2+ chelator ethylene glycol tetraacetic acid; neocortical Ca2+ levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca2+-containing postmitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca2+ levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca2+ homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

title: Brain region-specific altered expression and association of mitochondria-related genes in autism
abstract: BackgroundMitochondrial dysfunction (MtD) has been observed in approximately five percent of children with autism spectrum disorders (ASD). MtD could impair highly energy-dependent processes such as neurodevelopment, thereby contributing to autism. Most of the previous studies of MtD in autism have been restricted to the biomarkers of energy metabolism, while most of the genetic studies have been based on mutations in the mitochondrial DNA (mtDNA). Despite the mtDNA, most of the proteins essential for mitochondrial replication and function are encoded by the genomic DNA; so far, there have been very few studies of those genes. Therefore, we carried out a detailed study involving gene expression and genetic association studies of genes related to diverse mitochondrial functions.MethodsFor gene expression analysis, postmortem brain tissues (anterior cingulate gyrus (ACG), motor cortex (MC) and thalamus (THL)) from autism patients (n=8) and controls (n=10) were obtained from the Autism Tissue Program (Princeton, NJ, USA). Quantitative real-time PCR arrays were used to quantify the expression of 84 genes related to diverse functions of mitochondria, including biogenesis, transport, translocation and apoptosis. We used the delta delta Ct (∆∆Ct) method for quantification of gene expression. DNA samples from 841 Caucasian and 188 Japanese families were used in the association study of genes selected from the gene expression analysis. FBAT was used to examine genetic association with autism.ResultsSeveral genes showed brain region-specific expression alterations in autism patients compared to controls. Metaxin 2 (MTX2), neurofilament, light polypeptide (NEFL) and solute carrier family 25, member 27 (SLC25A27) showed consistently reduced expression in the ACG, MC and THL of autism patients. NEFL (P = 0.038; Z-score 2.066) and SLC25A27 (P = 0.046; Z-score 1.990) showed genetic association with autism in Caucasian and Japanese samples, respectively. The expression of DNAJC19, DNM1L, LRPPRC, SLC25A12, SLC25A14, SLC25A24 and TOMM20 were reduced in at least two of the brain regions of autism patients.ConclusionsOur study, though preliminary, brings to light some new genes associated with MtD in autism. If MtD is detected in early stages, treatment strategies aimed at reducing its impact may be adopted.

title: Downregulation of the Expression of Mitochondrial Electron Transport Complex Genes in Autism Brains
abstract: Mitochondrial dysfunction (MtD) and abnormal brain bioenergetics have been implicated in autism, suggesting possible candidate genes in the electron transport chain (ETC). We compared the expression of 84 ETC genes in the post‐mortem brains of autism patients and controls. Brain tissues from the anterior cingulate gyrus, motor cortex, and thalamus of autism patients (n = 8) and controls (n = 10) were obtained from Autism Tissue Program, USA. Quantitative real‐time PCR arrays were used to quantify gene expression. We observed reduced expression of several ETC genes in autism brains compared to controls. Eleven genes of Complex I, five genes each of Complex III and Complex IV, and seven genes of Complex V showed brain region‐specific reduced expression in autism. ATP5A1 (Complex V), ATP5G3 (Complex V) and NDUFA5 (Complex I) showed consistently reduced expression in all the brain regions of autism patients. Upon silencing ATP5A1, the expression of mitogen‐activated protein kinase 13 (MAPK13), a p38 MAPK responsive to stress stimuli, was upregulated in HEK 293 cells. This could have been induced by oxidative stress due to impaired ATP synthesis. We report new candidate genes involved in abnormal brain bioenergetics in autism, supporting the hypothesis that mitochondria, critical for neurodevelopment, may play a role in autism.

title: Brain Transcriptional and Epigenetic Associations with Autism
abstract: Autism is a common neurodevelopmental syndrome. Numerous rare genetic etiologies are reported; most cases are idiopathic.  To uncover important gene dysregulation in autism we analyzed carefully selected idiopathic autistic and control cerebellar and BA19 (occipital) brain tissues using high resolution whole genome gene expression and whole genome DNA methylation microarrays. No changes in DNA methylation were identified in autistic brain but gene expression abnormalities in two areas of metabolism were apparent: down-regulation of genes of mitochondrial oxidative phosphorylation and of protein translation. We also found associations between specific behavioral domains of autism and specific brain gene expression modules related to myelin/myelination, inflammation/immune response and purinergic signaling.  This work highlights two largely unrecognized molecular pathophysiological themes in autism and suggests differing molecular bases for autism behavioral endophenotypes.

title: Autism and lactic acidosis
abstract: Four patients are described who have two coexistent syndromes: the behavioral syndrome of autism and the biochemical syndrome of lactic acidosis. One of the four patients also had hyperuricemia and hyperuricosuria. These patients raise the possibility that one subgroup of the autism syndrome may be associated with inborn errors of carbohydrate metabolism.

title: Relative Carnitine Deficiency in Autism
abstract: A random retrospective chart review was conducted to document serum carnitine levels on 100 children with autism. Concurrently drawn serum pyruvate, lactate, ammonia, and alanine levels were also available in many of these children. Values of free and total carnitine (p < 0.001), and pyruvate (p=0.006) were significantly reduced while ammonia and alanine levels were considerably elevated (p < 0.001) in our autistic subjects. The relative carnitine deficiency in these patients, accompanied by slight elevations in lactate and significant elevations in alanine and ammonia levels, is suggestive of mild mitochondrial dysfunction. It is hypothesized that a mitochondrial defect may be the origin of the carnitine deficiency in these autistic children.

title: Mitochondrial dysfunction in autistic patients with 15q inverted duplication
abstract: Two autistic children with a chromosome 15q11‐q13 inverted duplication are presented. Both had uneventful perinatal courses, normal electroencephalogram and magnetic resonance imaging scans, moderate motor delay, lethargy, severe hypotonia, and modest lactic acidosis. Both had muscle mitochondrial enzyme assays that showed a pronounced mitochondrial hyperproliferation and a partial respiratory chain block most parsimoniously placed at the level of complex III, suggesting candidate gene loci for autism within the critical region may affect pathways influencing mitochondrial function.

title: Mitochondrial Dysfunction in Patients With Hypotonia, Epilepsy, Autism, and Developmental Delay: HEADD Syndrome
abstract: A group of 12 children clinically presenting with hypotonia, intractable epilepsy, autism, and developmental delay, who did not fall into previously described categories of mitochondrial encephalomyopathy, were evaluated for mitochondrial respiratory enzyme activity levels, mitochondrial DNA, and mitochondrial structural abnormalities. Reduced levels in specific respiratory activities were found solely in enzymes with subunits encoded by mitochondrial DNA in seven of eight biopsied skeletal muscle specimens evaluated. Five cases exhibited increased levels of large-scale mitochondrial DNA deletions, whereas pathogenic point mutations previously described in association with mitochondrial encephalomyopathies were not found. Mitochondrial structural abnormalities were present in three of four patients examined. Our findings suggest that mitochondrial dysfunction, including extensive abnormalities in specific enzyme activities, mitochondrial structure, and mitochondrial DNA integrity, may be present in children with a clinical constellation including hypotonia, epileptic seizures, autism, and developmental delay. The acronym HEADD is presented here to facilitate pursuit of mitochondrial defects in patients with this clinical constellation after other causes have been excluded.

title: Motor Matters: Tackling Heterogeneity of Parkinson’s Disease in Functional MRI Studies
abstract: To tackle the heterogeneity of Parkinson’s disease symptoms, most functional imaging studies tend to select a uniform group of subjects. We hypothesize that more profound considerations are needed to account for intra/inter-subject clinical variability and possibly for differing pathophysiological processes. Twelve patients were investigated using functional magnetic resonance imaging during visually-guided finger tapping. To account for disease heterogeneity, the motor score and individual symptom scores from the Unified Parkinson’s Disease Rating Scale (UPDRS-III) were utilized in the group-level model using two approaches either as the explanatory variable or as the effect of interest. Employment of the UPDRS-III score and symptom scores was systematically tested on the resulting group response to the levodopa challenge, which further accentuated the diversity of the diseased state of participants. Statistics revealed a bilateral group response to levodopa in the basal ganglia. Interestingly, systematic incorporation of individual motor aspects of the disease in the modelling amended the resulting activity patterns conspicuously, evidencing a manifold amount of explained variability by the particular score. In conclusion, the severity of clinical symptoms expressed in the UPDRS-III scores should be considered in the analysis to attain unbiased statistics, draw reliable conclusions and allow for comparisons between research groups studying Parkinson’s disease using functional magnetic resonance imaging.

title: The Heterogeneity of Early Parkinson’s Disease: A Cluster Analysis on Newly Diagnosed Untreated Patients
abstract: The variability in the clinical phenotype of Parkinson’s disease seems to suggest the existence of several subtypes of the disease. To test this hypothesis we performed a cluster analysis using data assessing both motor and non-motor symptoms in a large cohort of newly diagnosed untreated PD patients.  We collected data on demographic, motor, and the whole complex of non-motor symptoms from 100 consecutive newly diagnosed untreated outpatients. Statistical cluster analysis allowed the identification of different subgroups, which have been subsequently explored.  The data driven approach identified four distinct groups of patients, we have labeled: 1) Benign Pure Motor; 2) Benign mixed Motor-Non-Motor; 3) Non-Motor Dominant; and 4) Motor Dominant.  Our results confirmed the existence of different subgroups of early PD patients. Cluster analysis revealed the presence of distinct subtypes of patients profiled according to the relevance of both motor and non-motor symptoms. Identification of such subtypes may have important implications for generating pathogenetic hypotheses and therapeutic strategies.

title: Circulating interleukin‐10 and interleukin‐12 in Parkinson’s disease
abstract: Interleukin (IL)‐12 is a heterodimeric cytokine produced by activated blood monocytes, macrophages and glial cells. It enhances differentiation and proliferation of T cells and increases production of proinflammatory cytokines. IL‐10 is a pleiotropic cytokine produced by both lymphocytes and mononuclear phagocytes including microglia. Recent studies demonstrated the neuroprotective effect of IL‐10. There is little information about the involvement of IL‐12 or IL‐10 in the pathophysiology of Parkinson’s disease (PD).

title: Nitrated alpha‐synuclein‐activated microglial profiling for Parkinson’s disease
abstract: (2008) 104, 1504–1525.

title: Increased serum levels of soluble tumor necrosis factor-α receptor-1 in patients with Parkinson's disease
abstract: Parkinson's disease (PD) is characterized by a progressive and irreversible loss of dopaminergic neurons. Inflammatory mechanisms have been implied in the pathophysiology of PD. In this study, we assessed serum levels of TNF-alpha and the soluble forms of their receptors, sTNFR1 and sTNFR2, in 46 PD patients and 23 control subjects. Patients with PD had higher levels of sTNFR1 (p=0.048). The concentration of sTNFR1 and sTNFR2 correlated with age (p=0.006 and p=0.022, respectively). Higher levels of sTNFR1 were associated with later disease onset (p=0.048). These results corroborate the role of inflammatory events in the process of neurodegeneration in PD which can be of special relevance in the sporadic form of PD with later onset.

title: Serum inflammatory biomarkers in Parkinson's disease.
abstract: Numerous recent findings indicate the involvement of a neuroinflammatory reaction in the neurodegeneration in idiopathic Parkinson's disease (PD). We examined 29 consecutive patients with PD, ages 54-84 years, most of whom were moderately impaired (median UPDRS 19; Hoehn-Yahr 3; MMSE 28). A series of serum biomarkers were investigated, and their levels were correlated with the degree of the motor and cognitive impairment. There were no abnormalities of IL-6, acute phase proteins (C-reactive protein, serum amyloid A, alpha 1-antitrypsin, orosomucoid, ceruloplasmin, alpha 2-macroglobulin, transferrin, prealbumin) and factors of the complement system (C1q, C1-INH, C3, C4). A decrease in Mannan-binding lectin (MBL) levels was observed in six patients; an elevation of tumor necrosis factor-alpha (TNF-alpha) was found in 12 patients. No statistically significant correlation was found between the patient's clinical state (neuropsychologic and motor, as expressed by UPDRS III, Hoehn-Yahr, and MMSE) and the immunomarker changes. Our results indicate that the inflammatory process may be reflected in the serum; nevertheless, further research is needed to elucidate the possible clinical implications.

title: Interleukin-1 beta, interleukin-6, epidermal growth factor and transforming growth factor-alpha are elevated in the brain from parkinsonian patients.
abstract: Interleukin (IL)-1 beta, IL-6, epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) were measured for the first time in the brain (caudate nucleus, putamen and cerebral cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme immunoassays. The concentrations of IL-1 beta, IL-6, EGF, and TGF-alpha in the dopaminergic, striatal regions were significantly higher in parkinsonian patients than those in controls, whereas those in the cerebral cortex did not show significant differences between parkinsonian and control subjects. Since these cytokines and growth factors may play important roles as neurotrophic factors in the brain, the present results suggest that they may be produced as compensatory responses in the nigrostriatal dopaminergic regions in Parkinson's disease, and may be related, at least in part, to the process of neurodegeneration in Parkinson's disease.

title: Interleukin (IL)-1 beta, IL-2, IL-4, IL-6 and transforming growth factor-alpha levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's disease.
abstract: Interleukin (IL)-1 beta , IL-2, IL-4, IL-6, epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha were measured for the first time in ventricular cerebrospinal fluid (VCSF) from control non-parkinsonian patients, patients with juvenile parkinsonism (JP) and patients with Parkinson's disease (PD) by highly sensitive sandwich enzyme immunoassays. All cytokines were detectable in VCSF from control and parkinsonian patients, and the concentrations were much higher than those in lumbar CFS. The concentrations of IL-1 beta, IL-2, IL-4 and TGF-alpha in VCSF were higher in JP than those in controls (P < 0.05). In contrast, the concentrations of IL-2 and IL-6 in VCSF from patients with PD were higher than those from control patients (P < 0.05). These results agree with our previous reports, in which the cytokine levels were elevated in the striatal dopaminergic region of the brain from patients with PD. Since VCSF is produced in the ventricles, the alteration of cytokines in VCSF may reflect the changes of cytokines in the brain. Because cytokines play an important role as mitogens and neurotrophic factors in the brain, the increases in cytokines as a compensatory response may occur in the brain of patients of JP or PD during the progress of neurodegeneration. Increase in cytokines may contribute not only as a compensatory response but as a primary initiating trigger for the neurodegeneration.

title: Cytokines in Parkinson’s Disease
abstract: We found that in Parkinson’s disease (PD) the levels of various cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-2, IL-4, IL-6, epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β1] were significantly increased in the striatum (caudate and putamen) of the postmortem brain and in ventricular or spinal cerebrospinal fluid (VCSF, LCSF). Furthermore, the levels of the apoptosis-related proteins such as bcl-2 and soluble Fas (sFas) in the striatum were also elevated in PD. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonism mice, the levels of IL-1β in the striatum were significantly increased, but those of nerve growth factor (NGF) were significantly decreased, compared with control mice. In hemiparkinsonism rats produced by injection of 6-hydroxydopamine (6-OHDA) into one side of the median forebrain bundle, the levels of TNF-α in the 6-OHDA-treated side were increased in the striatum and substantia nigra, but not in the cerebral cortex, compared with those in the control side. Repeated administration of L-DOPA in the 6-OHDA-treated rats did not change the TNF-α levels in the control side and in the 6-OHDA-treated side in the substantia nigra, striatum, and cerebral cortex.

title: Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients.
abstract: Tumor necrosis factor-alpha (TNF-alpha), a glial-cell-related factor, was measured for the first time in the brain (striatum) and cerebrospinal fluid (CSF) from control and parkinsonian patients by a sensitive sandwich enzyme immunoassay. The concentrations of TNF-alpha in the brain and CSF were significantly higher in parkinsonian patients than those in controls. Since TNF-alpha is an important signal transducer of the immune system with cytotoxic and stimulator properties, these results suggest that an immune response may occur in the nigrostriatal dopaminergic regions in Parkinson's disease and that TNF-alpha may be related, at least in part, to the neuronal degeneration.

title: Impaired cytokine production by peripheral blood mononuclear cells and monocytes/macrophages in Parkinson's disease
abstract: Although the pathogenesis of Parkinson's disease (PD) is still unknown, several reports suggest the presence of immunological abnormalities in the patients with PD such as impaired T cell responses or cytokine production by the peripheral immune system.  In this study, we examined cytokine production by peripheral blood mononuclear cells (PBMC) and monocyte/macrophages (PBM) in the patients with idiopathic PD, using age‐related healthy donors as a normal control and cerebrovascular diseases (CVD) as a disease control.  Production of TNF‐α, IL‐1α, IL‐1β and IL‐6 by PBMC and TNFα by PBM were significantly lower in the patients with PD as compared to the control groups. IFN‐γ production by LPS‐stimulated PBMC in the patients with PD was also significantly lower than that in control groups. Cytokine production by PBMC from the patients with CVD who had a similar disability as the patient group was not significantly different from those in normal controls. Thus, impaired production of inflammatory cytokines may not be due to the mental and physical stress caused by their disability.  In the patients with PD, a significant negative correlation was noted in IL‐1α, IL‐1β and IL‐6 levels produced by LPS‐stimulated PBMC and Hoehn–Yahr disability score of the patients, suggesting that the impaired cytokine production may progress with disease progression. These abnormalities in cytokine production may not be primary but may affect the prognosis of PD.

title: Inflammation and infection in Parkinson's disease.
abstract: The hallmark of Parkinson's disease (PD) is a specific degeneration of dopaminergic neurons in the substantia nigra (SN). The cause of nigral dopaminergic neuronal cell death in PD and its underlying mechanisms remain elusive, however, involvement of inflammatory events has been postulated because inflammatory features have been described in the brain of PD patients. Some evidence also suggest that a possible deleterious effects of neuroinflammatory processes by infection in experimental models of neurodegenerative disease. In this review, we summarize and discuss the latest findings regarding inflammation in PD. Especially, we focused on the relationship between infection and PD.

title: Elevated levels of oxidized cholesterol metabolites in Lewy body disease brains accelerate α-synuclein fibrilization
abstract: Oxidative stress, inflammation and α-synuclein overexpression1 confer risk for development of α-synucleinopathies—neurodegenerative diseases that include Parkinson disease and Lewy body dementia2. Dopaminergic neurons undergo degeneration in these diseases and are particularly susceptible to oxidative stress because dopamine metabolism itself creates reactive oxygen species3. Intraneuronal deposition of α-synuclein as amyloid fibrils or Lewy bodies is the hallmark of these diseases2. Herein, we demonstrate that concentrations of oxidative cholesterol metabolites derived from reactive oxygen species are elevated in the cortices of individuals with Lewy body dementia relative to those of age-matched controls, and we show that these metabolites accelerate α-synuclein aggregation in vitro. The increase in the production of these cytotoxic cholesterol metabolites is also observed in a dopaminergic cell line that overexpresses α-synuclein4. By extension, these data lead to the hypothesis that oxidative stress produces cholesterol aldehydes that enable α-synuclein aggregation, leading to a pathologic cycle.

title: Oxidative damage in nucleic acids and Parkinson's disease
abstract: Oxidative DNA lesions, such as 8‐oxoguanine (8‐oxoG), accumulate in nuclear and mitochondrial genomes during aging, and such accumulation can increase dramatically in patients with Parkinson's disease (PD). To counteract oxidative damage to nucleic acids, human and rodents are equipped with three distinct enzymes. One of these, MTH1, hydrolyzes oxidized purine nucleoside triphosphates, such as 8‐oxo‐2′‐deoxyguanosine triphosphate and 2‐hydroxy‐2′‐deoxyadenosine triphosphate, to their monophosphate forms. The other two enzymes are 8‐oxoG DNA glycosylase encoded by the OGG1 gene and adenine/2‐hydroxyadenine DNA glycosylase encoded by the MUTYH gene. We have shown a significant increase in 8‐oxoG in mitochondrial DNA as well as an elevated expression of MTH1, OGG1, and MUTYH in nigrostriatal dopaminergic neurons of PD patients, suggesting that the buildup of these lesions may cause dopamine neuron loss. We established MTH1‐null mice and found that MTH1‐null fibroblasts were highly susceptible to cell death caused by H2O2 characterized by pyknosis and electron‐dense deposits in the mitochondria, and that this was accompanied by an ongoing accumulation of 8‐oxoG in nuclear and mitochondrial DNA. We also showed that MTH1‐null mice exhibited an increased accumulation of 8‐oxoG in striatal mitochondrial DNA, followed by more extreme neuronal dysfunction after 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine administration than that of wild‐type mice. In conclusion, oxidative damage in nucleic acids is likely to be a major risk factor for Parkinson's disease, indicating that a solid understanding of the defense mechanisms involved will enable us to develop new strategies for protecting the brain against oxidative stress.

title: Glutathione and Parkinson's disease: is this the elephant in the room?
abstract: At least 2 decades have past since the demonstration of a 40-50% deficit in total glutathione (GSH) levels in the substantia nigra in patients with Parkinson's disease (PD). The similar loss of GSH in the nigra in Incidental Lewy body disease, thought to be an early form of PD, indicates that this is one of the earliest derangements to occur in the pre-symptomatic stages of PD. Oxidative damage to lipids, protein and DNA in the nigra of PD patients is consistent with the loss of the antioxidant functions contributed by GSH. Past clinical trials that have used an antioxidant approach to treatment have used antioxidants that might substitute for GSH but these have shown modest to little benefit. More recent studies of the functions served by GSH in cells include in addition to its well-known participation in H(2)O(2) and toxin removal, such roles as modulation of protein function via thiolation which may control physiological and pathophysiological pathways to include DNA synthesis and repair, protein synthesis, amino acid transport, modulation of glutamate receptors and neurohormonal signaling. These multifunctional aspects to the workings of GSH in the cell would suggest that its loss perturbs many different processes and that replenishment and maintenance of GSH per se may be the best approach for preventing progressive damage from occurring. Despite this, few studies have been directed at specifically restoring GSH, although, as discussed herein, its unsanctioned use in PD is growing in popularity. This review will focus on glutathione in PD; the various functions carried out by glutathione and possible consequences of its depletion, as well as measures to elevate GSH in the CNS and its use in humans. Consideration of how the CNS generates and handles the substrates for GSH synthesis is also addressed with the view in mind that this may provide insights into control and maintenance of intracellular glutathione.

title: Parkinson's disease: A disorder due to nigral glutathione deficiency?
abstract: Amino acid analysis of autopsied human brain showed reduced glutathione (GSH) content significantly lower in the substantia nigra than in other brain regions. GSH was virtually absent in the nigra of patients with Parkinson's disease. Oxidative degradation of L-DOPA and dopamine in vivo may generate reactive oxygen species (hydrogen peroxide, superoxide, hydroxyl radical, or singlet oxygen) which can damage membranes and other cellular components. Since GSH is an important natural antioxidant, a deficiency of GSH in the substantia nigra could make this region vulnerable to oxidative injury. If confirmed, the hypothesis that loss of nigrostriatal dopaminergic neurons results from a regional GSH deficiency could have important therapeutic implications for the management and prevention of Parkinson's disease.

title: Idiopathic Parkinson's disease, progressive supranuclear palsy and glutathione metabolism in the substantia nigra of patients
abstract: Glutathione transferase activity and total glutathione (GSH) content were measured in several regions of autopsied brain from patients dying with idiopathic Parkinson's disease (PD) or progressive supranuclear palsy (PSP), and from control subjects. A significant deficiency of GSH was found in the substantia nigra, but not in 5 other brain regions of PD patients, nor in PSP patients' brains. Glutathione transferase activity was similar in the substantia nigra of PD, PSP and control patients. Since total GSH is consumed only by conjugation in detoxification processes, nigral GSH deficiency in PD patients implies continued local presence of a possible causative neurotoxin up to the time of death.

title: Reduced and oxidized glutathione in the substantia nigra of patients with Parkinson's disease
abstract: Reduced and oxidized glutathione concentrations in post-mortem brain tissue from the substantia nigra of control subjects and patients with neuropathologically confirmed Parkinson's disease were measured by a coulometric method using high-pressure liquid chromatography and electrochemical detection. Reduced glutathione concentrations were decreased in the substantia nigra of parkinsonian patients compared with controls. Differences in the concentration of oxidized glutathione and in the percentage of oxidized glutathione of the total glutathione were not observed between parkinsonian and control subjects. The finding that oxidized glutathione is not decreased in Parkinson's disease suggests that the decrease in reduced glutathione is not exclusively the consequence of neuronal loss in the substantia nigra but may indicate a state of oxidative stress.

title: A Generalised Increase in Protein Carbonyls in the Brain in Parkinson's but Not Incidental Lewy Body Disease
abstract: The degeneration of neurones in Parkinson's disease (PD) may involve oxidative stress. Previously, increased lipid peroxidation and oxidative DNA damage have been reported in parkinsonian substantia nigra. In the present study the protein carbonyl assay was used to assess oxidative protein damage in postmortem brain tissue from patients with PD and age‐matched controls. In brain areas associated with PD, such as substantia nigra, caudate nucleus, and putamen, there was a significant increase in carbonyl levels. However, increased carbonyl levels were also found in areas of the brain not thought to be affected in PD. This perhaps suggests that protein carbonyl formation is related to therapy with l‐DOPA, which can exert prooxidant properties in vitro. Consistent with this possibility, brain regions from individuals with incidental Lewy body disease (putative presymptomatic PD) showed no rise in carbonyls in any brain areas. Our data show that either oxidative protein damage occurs widely but late in PD brain, and/or that l‐DOPA treatment contributes to protein oxidation.

title: Increased Protein Oxidation in Human Substantia Nigra Pars Compacta in Comparison with Basal Ganglia and Prefrontal Cortex Measured with an Improved Dinitrophenylhydrazine Assay
abstract: The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4‐dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size‐dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.

title: Oxidative DNA Damage in the Parkinsonian Brain: An Apparent Selective Increase in 8‐Hydroxyguanine Levels in Substantia Nigra
abstract: Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8‐hydroxy‐2′‐deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8‐hydroxyguanine (8‐OHG) tended to be elevated and levels of 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8‐OHG in PD substantia nigra (p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8‐OHG is unlikely to be due to peroxynitrite (ONOO−) or hydroxyl radicals (OH•), or to be a prooxidant effect of treatment with l‐Dopa. However, some or all of the rise in 8‐OHG could be due to a change in 8‐OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.

title: Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease.
abstract: There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

title: Basal Lipid Peroxidation in Substantia Nigra Is Increased in Parkinson's Disease
abstract: Polyunsaturated fatty acid (PUFA) levels (an index of the amount of substrate available for lipid peroxidation) were measured in several brain regions from patients who died with Parkinson's disease and age‐matched control human postmortem brains. PUFA levels were reduced in parkinsonian substantia nigra compared to other brain regions and to control tissue. However, basal malondialdehyde (MDA; an intermediate in the lipid peroxidation process) levels were increased in parkinsonian nigra compared with other parkinsonian brain regions and control tissue. Expressing basal MDA levels in terms of PUFA content, the difference between parkinsonian and control substantia nigra was even more pronounced. Stimulating MDA production by incubating tissue with FeSO4 plus ascorbic acid, FeSO4 plus H2O2, or air alone produced lower MDA levels in the parkinsonian substantia nigra, probably reflecting the lower PUFA content. These results may indicate that an increased level of lipid peroxidation continues to occur in the parkinsonian nigra up to the time of death, perhaps because of continued exposure to excess free radicals derived from some endogenous or exogenous neurotoxic species.

title: Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions.
abstract: Aggregated alpha-synuclein proteins form brain lesions that are hallmarks of neurodegenerative synucleinopathies, and oxidative stress has been implicated in the pathogenesis of some of these disorders. Using antibodies to specific nitrated tyrosine residues in alpha-synuclein, we demonstrate extensive and widespread accumulations of nitrated alpha-synuclein in the signature inclusions of Parkinson's disease, dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and multiple system atrophy brains. We also show that nitrated alpha-synuclein is present in the major filamentous building blocks of these inclusions, as well as in the insoluble fractions of affected brain regions of synucleinopathies. The selective and specific nitration of alpha-synuclein in these disorders provides evidence to directly link oxidative and nitrative damage to the onset and progression of neurodegenerative synucleinopathies.

title: S-Nitrosylation of Parkin Regulates Ubiquitination and Compromises Parkin's Protective Function
abstract: Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.

title: Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity.
abstract: Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease.

title: (E)-4-hydroxy-2-nonenal may be involved in the pathogenesis of Parkinson's disease.
abstract: (E)-4-hydroxy-2-nonenal (HNE) is a toxic end-product of the free radical-stimulated peroxidation of phospholoipid-bound arachidonic acid in cell membranes. There is a growing body of evidence to suggest that free radicals may play an important role in the pathology of Parkinson's disease. HNE is highly electrophilic and is conjugated to reduced glutathione (GSH) by glutathione S-transferase. The depletion of GSH in the substantia nigra of Parkinson's patients and in the brainstem of mice treated with the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prompted this study on the concentrations of HNE in the cerebrospinal fluid (CSF) and plasma of Parkinson's patients and the brainstem of mice treated with MPTP. HNE was identified and quantitated by a highly specific and sensitive method based on the gas chromatography-negative-ion chemical ionisation mass spectrometry of the O-pentafluorobenzyl oxime derivative using 9D3-4-hydroxy-2-nonenal as an internal standard. The mean concentration of HNE in the CSF of patients with Parkinson's disease was 1.47+/-0.76 microM (mean+/-SD, n=10), while the concentration in the CSF of a group of control patients was 0.38+/-0.14 microM (n=10; p < .01). The mean concentration of HNE in the plasma of Parkinson's patients was 0.68+/-0.15 microM (n=20) and the concentration in the control group was 0.47+/-0 12 microM (n=20; p < .05). The mean peak concentration of HNE in the brainstem of mice after a single s.c. dose of MPTP (40 mg/kg) was 3.62+/-0.36 nM/g wet wt. (n=17) at 12 h while the control value was 0.45+/-0.05 nM/g wet wt. (n=20; p < .05). The GSH concentration in the brainstem of MPTP-treated mice at 24 h. was 0.65+/-0.03 microM/g wet wt. (n=14) and the control value was 1.25+/-0.03 microM/g wet wt. (n=20; p < .01). The corresponding concentration of GSH-HNE-conjugate at 24 h was 0.32+/-0.09 microM/g wet wt. (n=12) compared with a control value of 0.05+/-0.02 (n=16; p < .01). After treatment with alpha-tocopherol (2.35 g/kg s.c. daily x 3) the mean concentration of HNE 12 hr. after MPTP injection was 0.89+/-0.06 nM/g wet wt. (n=18). The HNE concentration in a group not treated with alpha-tocopherol prior to MPTP injection was 3.49+/-0.09 nM/g wet wt. (n=14; p < .05). The concentration of GSH in the mice pretreated with alpha-tocopherol before MPTP injection was 1.14+/-0.02 microM/g wet wt. (n=17) at 24 h compared to 0.61+/-0.02 microM/g wet wt. (n=14) in the untreated mice (p < .05). The direct injection of HNE (1, 10, 100, 1,000 microM) into the substantia nigra caused a dose dependent depletion of GSH in the brainstem of mice. The mean concentration of GSH 24 hr after the injection of 100 microM of HNE was 0.43+/-0.22 microM/g wet wt. (n=4) compared with a control value of 1.48+/-0.02 microM/g wet wt. (n=8; p < .05). The corresponding concentration of GSH-HNE-conjugate was 0.32+/-0.12 microM/g wet wt. (n=4) while the control value was 0.04+/-0.02 microM/g wet wt. (n=8). These data suggest that HNE may be a causative neurotoxin in Parkinson's disease and that HNE may also be involved in MPTP toxicity.

title: Systemic Increase of Oxidative Nucleic Acid Damage in Parkinson's Disease and Multiple System Atrophy
abstract: 8-hydroxy-2'-deoxyguanosine (8-OHdG) or 8-hydroxyguanosine (8-OHG), a product of oxidized DNA or RNA, is a good marker of oxidative cellular damage. In this study, we measured the 8-OHdG/8-OHG levels in the serum and cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) and multiple system atrophy (MSA). Compared to age-matched controls, the mean levels of serum 8-OHdG/8-OHG were significantly higher in PD (P < 0.0001). Although no gender differences were observed in the controls, the mean values of serum 8-OHdG/8-OHG were significantly higher in female PD cases (P < 0.005) than in male patients. 8-OHdG/8-OHG levels in CSF were also increased significantly in patients with PD and MSA, however, their relative values were generally much lower than those in the serum. Together with previous studies showing increased peripheral 8-OHdG levels in Alzheimer's disease and amyotrophic lateral sclerosis, the data presented here suggest that systemic DNA/RNA oxidation is commonly observed in neurodegenerative diseases. Our results also imply that female patients with PD show higher levels of oxidative stress, which may explain the faster progression of this disease in females.

title: Alteration of 8-hydroxyguanosine concentrations in the cerebrospinal fluid and serum from patients with Parkinson's disease
abstract: In order to investigate the possible role of oxidative RNA damage in the pathogenesis of Parkinson's disease (PD), the concentrations of the oxidative stress marker 8-hydroxyguanosine (8-OHG) were measured in the cerebrospinal fluid (CSF) and the serum of patients with PD and control subjects. The concentration of 8-OHG in CSF in PD patients was approximately three-fold that in controls (P < 0.001). The concentration of 8-OHG in CSF decreased significantly with the duration of disease (r(s) = -0.46, P < 0.05). However, the concentration of 8-OHG in serum was not significantly altered in PD patients compared to that in controls. In addition, the concentration of 8-OHG in CSF showed no correlation with that in serum in both the controls and PD patients suggesting that the 8-OHG concentrations in the CSF do not reflect those in serum and may be probably reflect those in brain tissue. These in vivo findings suggest a possible role of 8-OHG and increased oxidative RNA damage in the early stage of the development of PD.

title: Plasma and CSF markers of oxidative stress are increased in Parkinson's disease and influenced by antiparkinsonian medication
abstract: We determined systemic oxidative stress in Parkinson's disease (PD) patients, patients with other neurological diseases (OND) and healthy controls by measurement of in vitro lipoprotein oxidation and levels of hydro- and lipophilic antioxidants in plasma and cerebrospinal fluid (CSF). Additionally, we investigated the influence of levodopa (LD) and dopamine agonist therapy (DA) on the oxidative status in PD patients. We found increased oxidative stress, seen as higher levels of lipoprotein oxidation in plasma and CSF, decrease of plasma levels of protein sulfhydryl (SH) groups and lower CSF levels of alpha-tocopherol in PD patients compared to OND patients and controls. Levodopa treatment did not significantly change the plasma lipoprotein oxidation but LD monotherapy tended to result in an increase of autooxidation and in a decrease of plasma antioxidants with significance for ubiquinol-10. DA monotherapy was significantly associated with higher alpha-tocopherol levels. Patients with DA monotherapy or co-medication with DA showed a trend to lower lipoprotein oxidation. These data support the concept of oxidative stress as a factor in the pathogenesis of PD and might be an indicator of a potential prooxidative role of LD and a possible antioxidative effect of DA in PD treatment.

title: Redox status of plasma coenzyme Q10 indicates elevated systemic oxidative stress in Parkinson's disease
abstract: Oxidative stress is suggested to play an important role in the pathogenesis of Parkinson's disease (PD). However, no elevation of plasma oxidative stress marker has been reported. We measured percent content of the oxidized form of coenzyme Q10 in total coenzyme Q10 (%CoQ-10) because %CoQ-10 has been shown to be a sensitive marker of oxidative stress. A slight but significant elevation in %CoQ-10 was observed in PD patients when compared with age/gender-matched normal subjects, suggesting elevated systemic oxidative stress in PD patients.

title: Oxidative stress in peripheral blood mononuclear cells from patients with Parkinson's disease: Negative correlation with levodopa dosage
abstract: Oxidative stress, resulting from the imbalance between reactive oxygen species (ROS) formation and antioxidant defenses, plays a major role in the pathogenesis of Parkinson's disease (PD). However, the contribution of levodopa (LD) therapy to oxidative damage is still debated. We investigated oxidative stress in peripheral blood mononuclear cells (PBMCs) from LD-treated PD patients and healthy subjects. Increased ROS production associated with unaltered glutathione reductase activity was detected in PBMC from PD patients. LD daily dosage appeared to be inversely correlated with ROS levels and positively associated with GR activity, suggesting a protective role for LD on PBMCs redox status. Our data support the view of systemic oxidative stress involvement in PD and give further rationale for using PBMCs as an easily accessible ex-vivo dopaminergic model for exploring the biological effects of LD therapy.

title: Mitochondrial Complex I Deficiency in Parkinson's Disease
abstract: The structure and function of mitochondrial respiratory‐chain enzyme proteins were studied postmortem in the substantia nigra of nine patients with Parkinson's disease and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. NADH‐ubiquinone reductase (Complex I) and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of Complex I activity in the substantia nigra of patients with Parkinson's disease. This biochemical defect is the same as that produced in animal models of parkinsonism by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and adds further support to the proposition that Parkinson's disease may be due to an environmental toxin with action(s) similar to those of MPTP.

title: Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease.
abstract: Immunoblotting studies on mitochondria prepared from the striata of patients who died of Parkinson's disease were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with Parkinson's disease, the 30-, 25- and 24-kDa subunits of Complex I were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and Parkinson's disease. Deficiencies in Complex I subunits seem to be one of the most important clues to elucidate pathogenesis of Parkinson's disease.

title: Complex I deficiency in Parkinson's disease frontal cortex
abstract: A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinson's disease (PD) brain has identified loss of activity only in substantia nigra although loss of activity of this enzyme has been identified in a number of non-brain tissues. We investigated this paradox by studying complex I and other complexes of the mitochondrial electron transport chain in frontal cortex from PD and aged control brain using a variety of assay conditions and tissue preparations. We found increasingly significant losses of complex I activity in PD frontal cortex as increasingly pure mitochondria were studied. Complexes II, III, and IV were comparable in PD and controls. Inclusion of bovine serum albumin in the assay increased enzyme activity but lessened discrimination between PD and controls. Complex I deficiency in PD brain is not confined to substantia nigra. Methodological issues are critical in demonstrating this loss of activity.

title: Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease
abstract: Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and 1-methyl-4-phenylpyridine (MPP+), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.

title: Low platelet mitochondrial complex I and complex II/III activity in early untreated parkinson's disease
abstract: Following the discovery of inhibition of electron transport complex I by the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), which produces a parkinsonian syndrome in humans, monkeys, and mice, several laboratories have reported abnormalities of complex I and other electron transport complexes (ETCs) in various tissues from patients with Parkinson's disease (PD). Criticism of the significance of these findings in the etiology of PD has centered on whether drug treatments or the debilitation of the disease process itself produced the low ETC activities. We present results from a blinded study of platelet mitochondrial ETC activities in 18 early untreated PD patients and 18 age‐ and sex‐matched controls and in 13 spousal controls. Lower complex I activity in platelet mitochondria of PD patients was seen in early untreated disease and thus cannot be due to debilitation or drug therapy. Home environmental factors seem an unlikely explanation for the reduced complex I activity in PD patients but have not been excluded. Complex II/III activity was also reduced by 20% in PD compared with age‐/sex‐matched controls. The low complex I and II/III activities in platelet mitochondria appear to be related to the etiology of PD.

title: Parkinson's Disease Brain Mitochondrial Complex I Has Oxidatively Damaged Subunits and Is Functionally Impaired and Misassembled
abstract: Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II–V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

title: Respiratory-chain enzyme activities in isolated mitochondria of lymphocytes from patients with Parkinson's disease: preliminary study.
abstract: Evidence suggests that mitochondrial dysfunction stimulates the production of reactive oxygen species (ROS) that promote neural cell death in stroke and in Parkinson's disease. The sites of mitochondrial ROS production are not established but are generally believed to be located within the electron transport chain.



We studied the mitochondrial respiratory chain enzymes function from human circulating lymphocytes.



Open study.



Forty patients with Parkinson's disease (PD) with 30 age-matched control subjects were selected in this study. The patients had received no treatment before the study was conducted.



The data from patients and controls were compared using two-tailed student's t-test and values were expressed as means +/- standard deviation (SD).



Respiratory complex I + III and IV activities were significantly lower (P < 0.001) in patients than in control subjects.



The use of lymphocytes for investigating the respiratory chain enzymes provides an easy, noninvasive method to assess mitochondrial function in patients with PD. Furthermore, our study supports the hypothesis that a biochemical defect in the respiratory chain may be involved in the pathogenesis of PD.

title: An out‐of‐frame cytochrome b gene deletion from a patient with parkinsonism is associated with impaired complex III assembly and an increase in free radical production
abstract: We have isolated transmitochondrial cybrids containing a mitochondrial DNA cytochrome b 4–base pair deletion previously identified in a patient with parkinsonism. This presentation is in contrast to that of most patients with cytochrome b mutations, who present with exercise intolerance. Clones containing different levels of the cytochrome b 4–base pair deletion showed that high levels of the mutation were associated with a respiratory deficiency and a specific complex III defect. Newly synthesized full‐length cytochrome b was undetectable by metabolic labeling of mutant cells, and these cells were unable to grow in media that restricts proliferation of cells with defective oxidative phosphorylation. Steady state levels of some subunits previously found to be in close association with cytochrome b by crystallography and biochemical analysis (ie, Rieske [2Fe‐2S] protein and subunit VI) were drastically reduced in clones containing high levels of the mutation, whereas the reduction in the core‐1 subunit was milder. The absence of cytochrome b and complex III activity was also associated with increased hydrogen peroxide production. These findings, together with the variable tissue distribution of pathogenic mitochondrial DNA molecules, provide clues to the heterogeneous phenotypes associated with mitochondrial DNA mutations and establish a link between different forms of parkinsonism and oxidative phosphorylation defects.

title: Respiratory complex III is required to maintain complex I in mammalian mitochondria.
abstract: A puzzling observation in patients with oxidative phosphorylation (OXPHOS) deficiencies is the presence of combined enzyme complex defects associated with a genetic alteration in only one protein-coding gene. In particular, mutations in the mtDNA encoded cytochrome b gene are associated either with combined complex I+III deficiency or with only complex III deficiency. We have reproduced the combined complex I+III defect in mouse and human cultured cell models harboring cytochrome b mutations. In both, complex III assembly is impeded and causes a severe reduction in the amount of complex I, not observed when complex III activity was pharmacologically inhibited. Metabolic labeling in mouse cells revealed that complex I was assembled, although its stability was severely hampered. Conversely, complex III stability was not influenced by the absence of complex I. This structural dependence among complexes I and III was confirmed in a muscle biopsy of a patient harboring a nonsense cytochrome b mutation.

title: Parkinson's Disease and Brain Mitochondrial Dysfunction: A Functional Phosphorus Magnetic Resonance Spectroscopy Study
abstract: In spite of several evidences for a mitochondrial impairment in Parkinson's disease (PD), so far it has not been possible to show in vivo mitochondrial dysfunction in the human brain of PD patients. The authors used the high temporal and spatial resolution 31 phosphorus magnetic resonance spectroscopy (31P MRS) technique, which they have previously developed in normal subjects and in patients with mitochondrial diseases to study mitochondrial function by observing high-energy phosphates (HEPs) and intracellular pH (pH) in the visual cortex of 20 patients with PD and 20 normal subjects at rest, during, and after visual activation. In normal subjects, HEPs remained unchanged during activation, but rose significantly (by 16%) during recovery, and pH increased during visual activation with a slow return to rest values. In PD patients, HEPs were within the normal range at rest and did not change during activation, but fell significantly (by 36%) in the recovery period; pH did not reveal a homogeneous pattern with a wide spread of values. Energy unbalance under increased oxidative metabolism requirements, that is, the postactivation phase, discloses a mitochondrial dysfunction that is present in the brain of patients with PD even in the absence of overt clinical manifestations, as in the visual cortex. This is in agreement with our previous findings in patients with mitochondrial disease without clinical central nervous system (CNS) involvement. The heterogeneity of the physicochemical environment (i.e., pH) suggests various degrees of subclinical brain involvement in PD. The combined use of MRS and brain activation is fundamental for the study of brain energetics in patients with PD and may prove an important tool for diagnostic purposes and, possibly, to monitor therapeutic interventions.

title: Proton MR spectroscopy of the brain in 14 patients with Parkinson disease.
abstract: To determine whether the proton spectra from patients with clinically diagnosed Parkinson disease differ from the spectra of age-matched healthy subjects with respect to the major cerebral metabolite resonances as well as lactate.



Fourteen patients with Parkinson disease (38 to 81 years of age) and 13 healthy control subjects (37 to 81 years of age) were studied using image-guided, single-voxel (27-cm3 volume) proton MR spectroscopy of the occipital lobe.



The peak area ratios of N-acetyl aspartate to creatine and N-acetyl aspartate to choline for Parkinson patients did not show a statistically significant difference from the corresponding ratios for control subjects. There was a very significant increase in the ratio of lactate to N-acetyl aspartate for patients with Parkinson disease, with the greatest increase (threefold) manifested by the subgroup (n = 4) with dementia. The difference in N-acetyl aspartate to choline between women (n = 7) with Parkinson disease and healthy women (n = 9) approached significance. No dependence of the peak ratios on age, duration of Parkinson disease, or medication (L-dopa) regimen was found.



Preliminary results indicating an increase in cerebral lactate in patients with Parkinson disease support the hypothesis that Parkinson disease is a systemic disorder characterized by an impairment of oxidative energy metabolism. The larger increases for Parkinson patients with dementia may be diagnostically useful in assessing clinical course and in differentiating Parkinson disease from other causes of dementia. Additional studies are needed, though, to quantitate lactate changes and identify potential contributions from lipid resonances better.

title: Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis
abstract: Parkinson disease (PD) is associated with progressive loss of dopaminergic neurons in the substantia nigra, as well as with more-widespread neuronal changes that cause complex and variable motor and nonmotor symptoms. Recent rapid advances in PD genetics have revealed a prominent role for mitochondrial dysfunction in the pathogenesis of the disease, and the products of several PD-associated genes, including SNCA, Parkin, PINK1, DJ-1, LRRK2 and HTR2A, show a degree of localization to the mitochondria under certain conditions. Impaired mitochondrial function is likely to increase oxidative stress and might render cells more vulnerable to this and other related processes, including excitotoxicity. The mitochondria, therefore, represent a highly promising target for the development of disease biomarkers by use of genetic, biochemical and bioimaging approaches. Novel therapeutic interventions that modify mitochondrial function are currently under development, and a large phase III clinical trial is underway to examine whether high-dose oral coenzyme Q10 will slow disease progression. In this Review, we examine evidence for the roles of mitochondrial dysfunction and increased oxidative stress in the neuronal loss that leads to PD and discuss how this knowledge might further improve patient management and aid in the development of 'mitochondrial therapy' for PD.

title: Lower frequency of IL-17F sequence variant (His161Arg) in chronic fatigue syndrome patients.
abstract: Chronic fatigue syndrome (CFS) is characterized by immune dysfunctions including chronic immune activation, inflammation, and alteration of cytokine profiles. T helper 17 (Th17) cells belong to a recently identified subset of T helper cells, with crucial regulatory function in inflammatory and autoimmune processes. Th17 cells are implicated in allergic inflammation, intestinal diseases, central nervous system inflammation, disorders that may all contribute to the pathophysiology of CFS. IL-17F is one of the pro-inflammatory cytokines secreted by Th17 cells. We investigated the association between CFS and the frequency of rs763780, a C/T genetic polymorphism leading to His161Arg substitution in the IL-17F protein. The His161Arg variant (C allele) antagonizes the pro-inflammatory effects of the wild-type IL-17F. A significantly lower frequency of the C allele was observed in the CFS population, suggesting that the His161Arg variant may confer protection against the disease. These results suggest a role of Th17 cells in the pathogenesis of CFS.

title: Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells.
abstract: There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.

title: In chronic fatigue syndrome, the decreased levels of omega-3 poly-unsaturated fatty acids are related to lowered serum zinc and defects in T cell activation.
abstract: There is now evidence that major depression is accompanied by decreased levels of omega3 poly-unsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). There is a strong comorbidity between major depression and chronic fatigue syndrome (CFS). The present study has been carried out in order to examine PUFA levels in CFS. In twenty-two CFS patients and 12 normal controls we measured serum PUFA levels using gas chromatography and mass spectrometry. We found that CFS was accompanied by increased levels of omega6 PUFAs, i.e. linoleic acid and arachidonic acid (AA), and mono-unsaturated fatty acids (MUFAs), i.e. oleic acid. The EPA/AA and total omega3/omega6 ratios were significantly lower in CFS patients than in normal controls. The omega3/omega6 ratio was significantly and negatively correlated to the severity of illness and some items of the FibroFatigue scale, i.e. aches and pain, fatigue and failing memory. The severity of illness was significantly and positively correlated to linoleic and arachidonic acid, oleic acid, omega9 fatty acids and one of the saturated fatty acids, i.e. palmitic acid. In CFS subjects, we found significant positive correlations between the omega3/omega6 ratio and lowered serum zinc levels and the lowered mitogen-stimulated CD69 expression on CD3+, CD3+ CD4+, and CD3+ CD8+ T cells, which indicate defects in early T cell activation. The results of this study show that a decreased availability of omega3 PUFAs plays a role in the pathophysiology of CFS and is related to the immune pathophysiology of CFS. The results suggest that patients with CFS should respond favourably to treatment with--amongst other things--omega3 PUFAs, such as EPA and DHA.

title: Myalgic Encephalomyelitis (ME), Chronic Fatigue Syndrome (CFS), and Chronic Fatigue (CF) are distinguished accurately: Results of supervised learning techniques applied on clinical and inflammatory data
abstract: There is much debate on the diagnostic classification of Myalgic Encephalomyelitis (ME), Chronic Fatigue Syndrome (CFS) and chronic fatigue (CF). Post-exertional malaise (PEM) is stressed as a key feature. This study examines whether CF and CFS, with and without PEM, are distinct diagnostic categories. Fukuda's criteria were used to diagnose 144 patients with chronic fatigue and identify patients with CFS and CF, i.e. those not fulfilling the Fukuda's criteria. PEM was rated by means of a scale with defined scale steps between 0 and 6. CFS patients were divided into those with PEM lasting more than 24h (labeled: ME) and without PEM (labeled: CFS). The 12-item Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale was used to measure severity of illness. Plasma interleukin-1 (IL-1), tumor necrosis factor (TNF)α, and lysozyme, and serum neopterin were employed as external validating criteria. Using fatigue, a subjective feeling of infection and PEM we found that ME, CFS, and CF were distinct categories. Patients with ME had significantly higher scores on concentration difficulties and a subjective experience of infection, and higher levels of IL-1, TNFα, and neopterin than patients with CFS. These biomarkers were significantly higher in ME and CFS than in CF patients. PEM loaded highly on the first two factors subtracted from the data set, i.e. "malaise-sickness" and "malaise-hyperalgesia". Fukuda's criteria are adequate to make a distinction between ME/CFS and CF, but ME/CFS patients should be subdivided into ME (with PEM) and CFS (without PEM).

title: Chronic fatigue syndrome is associated with diminished intracellular perforin
abstract: Chronic fatigue syndrome (CFS) is an illness characterized by unexplained and prolonged fatigue that is often accompanied by abnormalities of immune, endocrine and cognitive functions. Diminished natural killer cell cytotoxicity (NKCC) is a frequently reported finding. However, the molecular basis of this defect of in vitro cytotoxicy has not been described. Perforin is a protein found within intracellular granules of NK and cytotoxic T cells and is a key factor in the lytic processes mediated by these cells. Quantitative fluorescence flow cytometry was used to the intracellular perforin content in CFS subjects and healthy controls. A significant reduction in the NK cell associated perforin levels in samples from CFS patients, compared to healthy controls, was observed. There was also an indication of a reduced perforin level within the cytotoxic T cells of CFS subjects, providing the first evidence, to our knowledge, to suggest a T cell associated cytotoxic deficit in CFS. Because perforin is important in immune surveillance and homeostasis of the immune system, its deficiency may prove to be an important factor in the pathogenesis of CFS and its analysis may prove useful as a biomarker in the study of CFS.

title: A formal analysis of cytokine networks in Chronic Fatigue Syndrome
abstract: Chronic Fatigue Syndrome (CFS) is a complex illness affecting 4 million Americans for which no characteristic lesion has been identified. Instead of searching for a deficiency in any single marker, we propose that CFS is associated with a profound imbalance in the regulation of immune function forcing a departure from standard pre-programmed responses. To identify these imbalances we apply network analysis to the co-expression of 16 cytokines in CFS subjects and healthy controls. Concentrations of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12, 13, 15, 17 and 23, IFN-γ, lymphotoxin-α (LT-α) and TNF-α were measured in the plasma of 40 female CFS and 59 case-matched controls. Cytokine co-expression networks were constructed from the pair-wise mutual information (MI) patterns found within each subject group. These networks differed in topology significantly more than expected by chance with the CFS network being more hub-like in design. Analysis of local modularity isolated statistically distinct cytokine communities recognizable as pre-programmed immune functional components. These showed highly attenuated Th1 and Th17 immune responses in CFS. High Th2 marker expression but weak interaction patterns pointed to an established Th2 inflammatory milieu. Similarly, altered associations in CFS provided indirect evidence of diminished NK cell responsiveness to IL-12 and LT-α stimulus. These observations are consistent with several processes active in latent viral infection and would not have been uncovered by assessing marker expression alone. Furthermore this analysis identifies key sub-networks such as IL-2:IFN-γ:TNF-α that might be targeted in restoring normal immune function.

title: The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group.
abstract: The complexities of the chronic fatigue syndrome and the methodologic problems associated with its study indicate the need for a comprehensive, systematic, and integrated approach to the evaluation, classification, and study of persons with this condition and other fatiguing illnesses. We propose a conceptual framework and a set of guidelines that provide such an approach. Our guidelines include recommendations for the clinical evaluation of fatigued persons, a revised case definition of the chronic fatigue syndrome, and a strategy for subgrouping fatigued persons in formal investigations.

title: Longitudinal investigation of natural killer cells and cytokines in chronic fatigue syndrome/myalgic encephalomyelitis
abstract: BackgroundChronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is an etiologically unexplained disorder characterised by irregularities in various aspects of the immunological function. Presently, it is unknown whether these immunological changes remain consistent over time. This study investigates Natural Killer (NK) cell cytotoxic activity, NK cell subsets (CD56brightCD16- and CD56dimCD16+) and cytokines, over the course of a12 month period in patients with CFS/ME.MethodsThe participants in the study comprised 65 (47.2 ± 11.5 years) CFS/ME participants and 21 (45.2 ±9.3 years) non-fatigued controls. Flow cytometry protocols were used to assess NK subsets and NK cytotoxic activity at various time points that included baseline (T1), 6 (T2) and 12 months (T3). Cytokine secretions were measured following mitogenic stimulation of peripheral blood mononuclear cells.ResultsNK cytotoxic activity was significantly decreased in the CFS/ME patients at T1, T2 and T3 compared to the non-fatigued group. Additionally, in comparison to the non-fatigued controls, the CFS/ME group had significantly lower numbers of CD56brightCD16- NK cells at both T1 and T2. Interestingly, following mitogenic stimulation, cytokine secretion revealed significant increases in IL-10, IFN-γ and TNF-α at T1 in the CFS/ME group. A significant decrease was observed at T2 in the CFS/ME group for IL-10 and IL-17A while at T3, IL-2 was increased in the CFS/ME group in comparison to the non-fatigued controls. Overall cytotoxic activity was significantly decreased at T3 compared to T1 and T2. CD56brightCD16- NK cells were much lower at T2 compared to T1 and T3. IL-10 and IL-17A secretion was elevated at T2 in comparison to T1 and T3.ConclusionThese results confirm decreases in immune function in CFS/ME patients, suggesting an increased susceptibility to viral and other infections. Furthermore, NK cytotoxic activity may be a suitable biomarker for diagnosing CFS/ME as it was consistently decreased during the course of the 12 months study.

title: Immunological abnormalities as potential biomarkers in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis
abstract: BackgroundChronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is characterised by severe prolonged fatigue, and decreases in cognition and other physiological functions, resulting in severe loss of quality of life, difficult clinical management and high costs to the health care system. To date there is no proven pathomechanism to satisfactorily explain this disorder. Studies have identified abnormalities in immune function but these data are inconsistent. We investigated the profile of markers of immune function (including novel markers) in CFS/ME patients.MethodsWe included 95 CFS/ME patients and 50 healthy controls. All participants were assessed on natural killer (NK) and CD8+T cell cytotoxic activities, Th1 and Th2 cytokine profile of CD4+T cells, expression of vasoactive intestinal peptide receptor 2 (VPACR2), levels of NK phenotypes (CD56bright and CD56dim) and regulatory T cells expressing FoxP3 transcription factor.ResultsCompared to healthy individuals, CFS/ME patients displayed significant increases in IL-10, IFN-γ, TNF-α, CD4+CD25+ T cells, FoxP3 and VPACR2 expression. Cytotoxic activity of NK and CD8+T cells and NK phenotypes, in particular the CD56bright NK cells were significantly decreased in CFS/ME patients. Additionally granzyme A and granzyme K expression were reduced while expression levels of perforin were significantly increased in the CFS/ME population relative to the control population. These data suggest significant dysregulation of the immune system in CFS/ME patients.ConclusionsOur study found immunological abnormalities which may serve as biomarkers in CFS/ME patients with potential for an application as a diagnostic tool.

title: Xenotropic murine leukemia virus-related virus-associated chronic fatigue syndrome reveals a distinct inflammatory signature.
abstract: The recent identification of xenotropic murine leukemia virus-related virus (XMRV) in the blood of patients with chronic fatigue syndrome (CFS) establishes that a retrovirus may play a role in the pathology in this disease. Knowledge of the immune response might lead to a better understanding of the role XMRV plays in this syndrome. Our objective was to investigate the cytokine and chemokine response in XMRV-associated CFS.



Using Luminex multi-analyte profiling technology, we measured cytokine and chemokine values in the plasma of XMRV-infected CFS patients and compared these data to those of healthy controls. Analysis was performed using the Gene Expression Pattern Analysis Suite and the Random Forest tree classification algorithm.



This study identifies a signature of 10 cytokines and chemokines which correctly identifies XMRV/CFS patients with 93% specificity and 96% sensitivity.



These data show, for the first time, an immunological pattern associated with XMRV/CFS.

title: TNF-alpha and chronic fatigue syndrome.
abstract: Based upon the clinical presentation of chronic fatigue syndrome (CFS), we hypothesized that proinflammatory cytokines may play a role in the pathogenesis of the disease. We therefore undertook a retrospective cross-sectional study to examine the role of TNF-alpha in patients with CFS. Our results suggest a significant increase serum TNF-alpha in patients with CFS (P<0.0001) compared to non-CFS controls. This study supports the further examination of the role of proinflammatory mediators in CFS. Furthermore, the clinical testing of TNF-alpha blockers and other antiinflammatory agents for the treatment of this disease is warranted.

title: Chronic fatigue syndrome: identification of distinct subgroups on the basis of allergy and psychologic variables.
abstract: We investigated a role for allergic inflammation and psychologic parameters in the development of chronic fatigue syndrome (CFS).



The design was a comparison between subjects with CFS and age- and sex-matched control cohorts. Studies were performed on CFS subjects (n = 18) and control cohorts consisting of normal subjects (n = 11), allergic subjects (n = 14), and individuals with primary depression (n = 12). We quantified cytokines at baseline as cell-associated immunoreactive peptides and as transcripts evaluated by means of semiquantitative RNA-based polymerase chain reactions. Psychologic evaluations included administration of the Diagnostic Interview Schedule, the Structured Clinical Interview, and the Symptom Checklist 90-Revised.



Increases in tumor necrosis factor (TNF)-alpha were identified in individual subjects with CFS (50.1 +/- 14.4 pg TNF-alpha per 10(7) peripheral blood mononuclear cells [PBMCs]; mean +/- SEM) and allergic subjects (41.6 +/- 7.6) in comparison with normal subjects (13.1 +/- 8.8) (P < .01 and P < .05, respectively). Similar trends were observed for interferon (IFN)-alpha in allergic subjects (3.0 +/- 1.7 pg/10(7) PBMCs) and subjects with CFS (6.4 +/- 3.4) compared with normal subjects (1.9 +/- 1.4). A significant increase (P < .05) in TNF-alpha transcripts was demonstrated between subjects with CFS and depressed subjects. In contrast to these proinflammatory cytokines, both subjects with CFS (2.6 +/- 1.8 pg/10(7) PBMCs) and allergic subjects (3.4 +/- 2.8) were associated with a statistically significant (P < .01) decrease in IL-10 concentrations compared with normal subjects (60.2 +/- 18.2). As shown in other studies, most of our subjects with CFS were allergic (15 of 18) and therefore presumably demonstrated cytokine gene activation on that basis. The seasonal exacerbation of allergy was associated with a further increase in cellular IFN-alpha (from 2.1 +/- 1.2 to 14.2 +/- 4.5 pg/107 PBMCs; P < .05) but no further modulation of TNF-alpha or IL-10. Similarly, self-reported exacerbations of CFS were associated with a further increase in IFN-alpha (from 2.5 +/- 1.0 to 21.9 +/- 7.8; P < .05) and occurred at times of seasonal exposures to allergens. This linkage does not permit making any definitive conclusions regarding a causative influence of either seasonal allergies or the increase in cellular IFN-alpha with the increase in CFS symptoms. The close association between atopy and CFS led us to speculate that CFS may arise from an abnormal psychologic response to the disordered expression of these proinflammatory and antiinflammatory cytokines. Psychologic variables were predictive of immune status within the CFS sample (65.9% of the variance in immune status; F (3,10) = 6.44, P < .05). Specifically, the absence of a personality disorder but greater endorsement of global psychiatric symptoms was predictive of immune activation.



Most of our subjects with CFS were allergic, and the CFS and allergy cohorts were similar in terms of their immune status. However, the CFS subjects could be discriminated by the distinct psychologic profiles among subjects with and without immune activation. We propose that in at least a large subgroup of subjects with CFS who had allergies, the concomitant influences of immune activation brought on by allergic inflammation in an individual with the appropriate psychologic profile may interact to produce the symptoms of CFS. In a psychologically predisposed individual, symptoms associated with allergic inflammation are recognized as illness.

title: Dysregulated expression of tumor necrosis factor in chronic fatigue syndrome: interrelations with cellular sources and patterns of soluble immune mediator expression.
abstract: Among a group of 70 individuals who met the criteria established by the Centers for Disease Control and Prevention (Atlanta) for chronic fatigue syndrome (CFS), 12%-28% had serum levels exceeding 95% of control values for tumor necrosis factor (TNF) alpha, TNF-beta, interleukin (IL) 1 alpha, IL-2, soluble IL-2 receptor (sIL-2R), or neopterin; overall, 60% of patients had elevated levels of one or more of the nine soluble immune mediators tested. Nevertheless, only the distributions for circulating levels of TNF-alpha and TNF-beta differed significantly in the two populations. In patients with CFS--but not in controls--serum levels of TNF-alpha, IL-1 alpha, IL-4, and sIL-2R correlated significantly with one another and (in the 10 cases analyzed) with relative amounts (as compared to beta-globin or beta-actin) of the only mRNAs detectable by reverse transcriptase-coupled polymerase chain reaction in peripheral-blood mononuclear cells: TNF-beta, unspliced and spliced; IL-1 beta, lymphocyte fraction; and IL-6 (in order of appearance). These findings point to polycellular activation and may be relevant to the etiology and nosology of CFS.

title: Immune Abnormalities in Patients Meeting New Diagnostic Criteria for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis
abstract: Immunological abnormalities have been identified in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients fulfilling the 1994 Centers for Disease Control diagnostic criteria. Significant developments have been made to diagnostic criteria, but potential immunological markers have not been assessed in patients fulfilling these latest clinical requirements. Therefore, this study evaluated immunological parameters in patients that also fulfill the latest diagnostic criteria available known as the International Consensus Criteria.  The Immunological investigations including Natural Killer cell activity and phenotyping studies for dendritic cells, neutrophils, B cells and regulatory T cells were performed on whole blood samples collected from all participants using flow cytometric protocols. The physical functioning of all participants was also evaluated using scores from the Short Form Health Survey, and the World Health Organization Disability Adjustment Schedule. Results were compared according 1994 Centers of Disease Control and Prevention defined patients, and International Consensus Criteria defined patients, and healthy controls.  Natural killer cell activity was consistently and significantly decreased, and regulatory T cells were significantly increased in both patient groups compared to healthy controls. Differences were found in human neutraphil antigens and expression of natural killer cell receptors between patient groups. Highly significant correlations were also found between physical status and some immune parameters in International Consensus Criteria defined patients.  This preliminary investigation on different diagnostic criteria suggests that the International Consensus Criteria may be more effective a detecting salient differences in the immune system.

title: Evidence for the Presence of Immune Dysfunction in Chronic Fatigue Syndrome
abstract: Chronic fatigue syndrome is a medically unexplained ailment characterized by new onset of fatigue accompanied by rheumatological, infectious, and neuropsychiatric symptoms. Because the ailment often begins suddenly with a flu-like presentation, early pathophysiological ideas as to cause included

title: Brain dysfunction as one cause of CFS symptoms including difficulty with attention and concentration
abstract: We have been able to reduce substantially patient pool heterogeneity by identifying phenotypic markers that allow the researcher to stratify chronic fatigue syndrome (CFS) patients into subgroups. To date, we have shown that stratifying based on the presence or absence of comorbid psychiatric diagnosis leads to a group with evidence of neurological dysfunction across a number of spheres. We have also found that stratifying based on the presence or absence of comorbid fibromyalgia leads to information that would not have been found on analyzing the entire, unstratified patient group. Objective evidence of orthostatic intolerance (OI) may be another important variable for stratification and may define a group with episodic cerebral hypoxia leading to symptoms. We hope that this review will encourage other researchers to collect data on discrete phenotypes in CFS to allow this work to continue more broadly. Finding subgroups of CFS suggests different underlying pathophysiological processes responsible for the symptoms seen. Understanding those processes is the first step toward developing discrete treatments for each.

title: Chronic fatigue syndrome, the immune system and viral infection
abstract: The chronic fatigue syndrome (CFS), as defined by recent criteria, is a heterogeneous disorder with a common set of symptoms that often either follows a viral infection or a period of stress. Despite many years of intense investigation there is little consensus on the presence, nature and degree of immune dysfunction in this condition. However, slightly increased parameters of inflammation and pro-inflammatory cytokines such as interleukin (IL) 1, IL6 and tumour necrosis factor (TNF) α are likely present. Additionally, impaired natural killer cell function appears evident. Alterations in T cell numbers have been described by some and not others. While the prevalence of positive serology for the common herpes viruses appears no different from healthy controls, there is some evidence of viral persistence and inadequate containment of viral replication. The ability of certain herpes viruses to impair the development of T cell memory may explain this viral persistence and the continuation of symptoms. New therapies based on this understanding are more likely to produce benefit than current methods.

title: Is chronic fatigue syndrome associated with platelet activation?
abstract: Chronic fatigue syndrome (CFS) is a debilitating condition that has no known aetiology or pathophysiology. Recent investigations by other workers have suggested that individuals with CFS may have a hypercoagulable state. This study investigated various aspects of platelet activation and function in 17 patients with CFS and in 16 age-matched and sex-matched healthy controls. Platelet aggregation, platelet volume and coagulation tests were performed. Platelet aggregation was investigated by means of the photometric changes using citrated platelet-rich plasma, whole blood aggregation was calculated as the percentage fall in single platelet counts and the coagulation tests were performed on an automatic microcentrifugal analyser.A trend was observed for the patients to have lower aggregation results and a reduced mean platelet volume. However, this only reached statistical significance for one result; the rate of the aggregation slope by 1.0 μg/ml collagen [CFS patients, 18 (9–28) versus controls, 32.5 (19–36); Mann–Whitney U test, P = 0.029]. No significant differences were found for any of the measurements of coagulation.These results are in contrast to previously reported findings. However, due to the heterogeneous nature of the disease, and the resulting lifestyles of the patients, caution should be taken when comparing one group of patients with another. Nevertheless, we certainly found no evidence of increased platelet activation or of a hypercoagulable state in patients with CFS and, on the basis of these results, anti-platelet or anti-coagulant therapy is not warranted.

title: Post-infective and chronic fatigue syndromes precipitated by viral and non-viral pathogens: prospective cohort study
abstract: To delineate the risk factors, symptom patterns, and longitudinal course of prolonged illnesses after a variety of acute infections.  Prospective cohort study following patients from the time of acute infection with Epstein-Barr virus (glandular fever), Coxiella burnetii (Q fever), or Ross River virus (epidemic polyarthritis).  The region surrounding the township of Dubbo in rural Australia, encompassing a 200 km geographical radius and 104 400 residents. Participants 253 patients enrolled and followed at regular intervals over 12 months by self report, structured interview, and clinical assessment. Outcome measures Detailed medical, psychiatric, and laboratory evaluations at six months to apply diagnostic criteria for chronic fatigue syndrome. Premorbid and intercurrent illness characteristics recorded to define risk factors for chronic fatigue syndrome. Self reported illness phenotypes compared between infective groups.  Prolonged illness characterised by disabling fatigue, musculoskeletal pain, neurocognitive difficulties, and mood disturbance was evident in 29 (12%) of 253 participants at six months, of whom 28 (11%) met the diagnostic criteria for chronic fatigue syndrome. This post-infective fatigue syndrome phenotype was stereotyped and occurred at a similar incidence after each infection. The syndrome was predicted largely by the severity of the acute illness rather than by demographic, psychological, or microbiological factors.  A relatively uniform post-infective fatigue syndrome persists in a significant minority of patients for six months or more after clinical infection with several different viral and non-viral micro-organisms. Post-infective fatigue syndrome is a valid illness model for investigating one pathophysiological pathway to chronic fatigue syndrome.

title: Implementing cognitive behavioral therapy for chronic fatigue syndrome in a mental health center: a benchmarking evaluation.
abstract: This study evaluated the success of implementing cognitive behavioral therapy (CBT) for chronic fatigue syndrome (CFS) in a representative clinical practice setting and compared the patient outcomes with those of previously published randomized controlled trials (RCTs) of CBT for CFS.



The implementation interventions were the following: spreading information about the new treatment setting to general practitioners and CFS patients; training mental health center (MHC) therapists in CBT for CFS; and organizing changes in the MHC patient workflow. Patient outcomes were documented with validated self-report measures of fatigue and physical functioning before and after treatment. The comparison of the treatment results with RCT results was done following the benchmark strategy.



One-hundred forty-three CFS patients were referred to the MHC, of whom 112 started treatment. The implementation was largely successful, but a weak point was the fact that 32% of all referred patients dropped out shortly after or even before starting treatment. Treatment effect sizes were in the range of those found in the benchmark studies.



CBT for CFS can successfully be implemented in an MHC. Treatment results were acceptable, but the relatively large early dropout of patients needs attention.

title: A Report–Chronic Fatigue Syndrome: Guidelines for Research
abstract: Authors Dr M C Sharpe MRCPsych Clinical Lecturer, Department of Psychiatry, University of Oxford Dr L C Archard PhD Senior Lecturer, Department of Biochemistry, Charing Cross and Westminster Medical School, London Professor J E Banatvala MD Professor of Virology, Department of Virology, St Thomas' Hospital Medical School, London Dr L K Borysiewicz FRCP Wellcome Trust Senior Lecturer, Department of Medicine, University of Cambridge Professor A W Clare MD Clinical Professor of Psychiatry, Trinity College, Dublin Dr A David MRCPsych MRC Training Fellow, Institute of Psychiatry, London Professor R H T Edwards FRCP Professor of Medicine, Department of Medicine, University of Liverpool Dr K E H Hawton DM Consultant Psychiatrist, Department of Psychiatry, University of Oxford Professor H P Lambert MD Emeritus Professor of Microbial Disease, St George's Hospital Medical School, London Dr R J M Lane MD Consultant Neurologist, Regional Neurosciences Centre, Charing Cross Hospital, London Dr E M McDonald MRCPsych Research Psychiatrist, Institute of Psychiatry, London Professor J F Mowbray FRCP Immunopathology, Department oflmmunopathology, St Mary's Hospital Medical School, London Dr D J Pearson FRCP Senior Lecturer, Department of Medicine, University of Manchester Dr TEA Peto MRCP Consultant Physician, Department of Infectious Diseases, John Radcliffe Hospital, Oxford Dr V R Preedy PhD Research Biochemist, Department of Biochemistry, King's College Hospital, London Dr A P Smith PhD Research Psychologist, Department of Psychology, University of Sussex Dr D G Smith MB General Practitioner, Horndonon-the-Hill, Essex Dr D J Taylor DPhil MRC Staff Scientist, MRC Biochemical and Clinical Magnetic Resonance Unit, John Radcliffe Hospital, Oxford Dr D A J Tyrrell MD Director, MRC Common Cold Unit, Harvard Hospital, Salisbury Dr S Wessely MRCPsych Wellcome Training Fellow in Epidemiology, Institute of Psychiatry, London Dr P D White MRCPsych Senior Lecturer, Department of Psychological Medicine, St Bartholomew's Hospital, London Other signatories who contributed to the guidelines but who were unable to attend the meeting Professor P 0 Behan MD Professor of Neurology, Department of Neurology, University of Glasgow Dr F Clifford Rose FRCP Director, Academic Unit of Neuroscience, Charing Cross and Westminster Medical School, London Professor T J Peters FRCP Professor of Clinical Biochemistry, Department of Biochemistry, King's College Hospital, London Dr P G Wallace MRCGP Head of Research Unit, Department of General Practice, St Mary's Hospital Medical School, London Professor D A Warrell FRCP Professor of Tropical Medicine and Infectious Diseases, Nuffield Department of Medicine, John Radcliffe Hospital, Oxford Dr D J M Wright MD Consultant Microbiologist, Department of Microbiology, Charing Cross Hospital, London

title: The Sickness Impact Profile: Development and Final Revision of a Health Status Measure
abstract: The final development of the Sickness Impact Profile (SIP), a behaviorally based measure of health status, is presented. A large field trial on a random sample of prepaid group practice enrollees and smaller trials on samples of patients with hyperthyroidism, rheumatoid arthritis and hip replacements were undertaken to assess reliability and validity of the SIP and provide data for category and item analyses. Test-retest reliability (r = 0.92) and internal consistency (r = 0.94) were high. Convergent and discriminant validity was evaluated using the multitrait–multimethod technique. Clinical validity was assessed by determining the relationship between clinical measures of disease and the SIP scores. The relationship between the SIP and criterion measures were moderate to high and in the direction hypotheszed. A technique for describing and assessing similarities and differences among groups was developed using profile and pattern analysis. The final SIP contains 136 items in 12 categories. Overall, category, and dimension scores may be calculated.

title: Longitudinal MRI shows no cerebral abnormality in chronic fatigue syndrome.
abstract: MRI has previously provided conflicting results when used to search for brain abnormalities in sufferers of chronic fatigue syndrome (CFS). Eighteen CFS patients and nine healthy volunteers each underwent MRI on two occasions, one year apart. The resulting images were examined for abnormalities in brain atrophy, deep white matter hyperintensities (WMH) and cerebral blood and cerebrospinal fluid (CSF) flow. Mean proportionate CSF volume was not significantly different between subject groups. All participants showed a slight increase in CSF between scans, but no significant difference was found between those with CFS and those without. Between-group comparisons of ventricular volume revealed no significant differences at study commencement and no significant change over the year. No significant inter-group differences were found for any of the cerebral blood and CSF flow parameters. Low levels of WMH were found in all participants. Objective scoring of WMH using Scheltens' scale revealed no change in summary components (prosencephalic deep white matter hyperintensities, basal ganglia hyperintensities and infratentorial hyperintensities) or in individual component variables between the baseline and 1 year follow-up scans. No abnormal patterns in rate and extent of brain atrophy, ventricle volume, white matter lesions, cerebral blood flow or aqueductal CSF flow were detected in the CFS population. These results throw open the debate into whether MRI scanning can reveal diagnostic signs of CFS and clinically questions the diagnoses of CFS made on the basis of previous research conclusions.

title: Mitochondrial enzymes discriminate between mitochondrial disorders and chronic fatigue syndrome.
abstract: We studied the extent of mitochondrial involvement in chronic fatigue syndrome (CFS) and investigated whether measurement of mitochondrial respiratory chain complex (RCC) activities discriminates between CFS and mitochondrial disorders. Mitochondrial content was decreased in CFS compared to healthy controls, whereas RCC activities corrected for mitochondrial content were not. Conversely, mitochondrial content did not discriminate between CFS and two groups of mitochondrial disorders, whereas ATP production rate and complex I, III and IV activity did, all with higher activities in CFS. We conclude that the ATP production rate and RCC activities can reliably discriminate between mitochondrial disorders and CFS.

title: Chronic fatigue syndrome is accompanied by an IgM-related immune response directed against neopitopes formed by oxidative or nitrosative damage to lipids and proteins.
abstract: There is now some evidence that chronic fatigue syndrome (CFS) is accompanied by signs of oxidative stress and by a decreased antioxidant status. The aim of the present study was to examine whether CFS is accompanied by an immune response to neoepitopes of a variety of modified lipids and proteins indicating damage caused by oxidative and nitrosative stress. Toward this end we examined serum antibodies to fatty acids (oleic, palmitic and myristic acid), by-products of lipid peroxidation, i.e. azelaic acid and malondialdehyde (MDA), acetylcholine, S-farnesyl-L-cysteine, and N-oxide modified amino-acids in 14 patients with CFS, 14 subjects with partial CFS and 11 normal controls. We found that the prevalences and mean values for the serum IgM levels directed against oleic, palmitic and myristic acid, MDA, azelaic acid, S-farnesyl-L-cysteine, and the N-oxide derivates, nitro-tyrosine, nitro-phenylalanine, nitro-arginine, nitro-tryptophan, and nitro-cysteinyl were significantly greater in CFS patients than in normal controls, whereas patients with partial CFS took up an intermediate position. There were significant and positive correlations between the serum IgM levels directed against fatty acids, MDA and azelaic acid and the above N-oxide-derivates and the severity of illness (as measured by the FibroFatigue scale) and symptoms, such as aches and pain, muscular tension and fatigue. The results show that CFS is characterized by an IgM-related immune response directed against disrupted lipid membrane components, by-products of lipid peroxidation, S-farnesyl-L-cysteine, and NO-modified amino-acids, which are normally not detected by the immune system but due to oxidative and nitrosative damage have become immunogenic.

title: Increased 8-hydroxy-deoxyguanosine, a marker of oxidative damage to DNA, in major depression and myalgic encephalomyelitis / chronic fatigue syndrome.
abstract: There is now evidence that major depression and myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) are accompanied by partially overlapping pathophysiological mechanisms, i.e. activation of various inflammatory and oxidative & nitrosative (IO&NS) pathways.



The aim of the present study was to examine the urinary excretion of 8-hydroxy-deoxyguanosine (8-OhdG), a marker of oxidative damage to DNA, in depression; ME/CFS; and depression and ME/CFS.



Toward this end, morning urine was sampled for the assays of 8-OHdG and creatinine, in 44 patients with ME/CFS; 25 with major depression; 23 with depression and ME/CFS; and 17 normal controls. Severity of fatigue and somatic symptoms was measured by means of the Fibromyalgia and CFS Rating (FF) scale.



We found that 49.0% of the variance in the urinary excretion of 8-OHdG was predicted by the regression on creatinine. Consequently, the urinary 8-OHdG excretion should be expressed as the residualized 8-OHdG values after partialling out the effects of creatinine and not by computing the 8-OHdG / creatinine ratio. We found that the residualized urinary excretion of 8-OHdG (adjusted for creatinine) was significantly higher in patients with depression and ME/CFS than in normal controls and all other patients. In the patient group, there were significant correlations between the urinary 8-OHdG and the total score on the FF scale and sadness and flu-like malaise.



The findings show increased oxidatively generated DNA damage in patients with major depression and ME/CFS and, therefore, further extent the role played by IO&NS pathways in the pathophysiology of both disorders. Since oxidatively damage to DNA is a risk factor for atherosclerosis and neurodegeneration, our results also explain previous findings on increased cardiovascular morbidity in depression and ME/CFS, and neurodegenerative processes in depression.

title: Coenzyme Q10 deficiency in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is related to fatigue, autonomic and neurocognitive symptoms and is another risk factor explaining the early mortality in ME/CFS due to cardiovascular disorder.
abstract: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a medical illness characterized by disorders in inflammatory and oxidative and nitrosative (IO&NS) pathways.



This paper examines the role of Coenzyme Q10 (CoQ10), a mitochondrial nutrient which acts as an essential cofactor for the production of ATP in mitochondria and which displays significant antioxidant activities. Plasma CoQ10 has been assayed in 58 patients with ME/CFS and in 22 normal controls; the relationships between CoQ10 and the severity of ME/CFS as measured by means of the FibroFatigue (FF) scale were measured.



Plasma CoQ10 was significantly (p=0.00001) lower in ME/CFS patients than in normal controls. Up to 44.8% of patients with ME/CFS had values beneath the lowest plasma CoQ10 value detected in the normal controls, i.e. 490 microg/L. In ME/CFS, there were significant and inverse relationships between CoQ10 and the total score on the FF scale, fatigue and autonomic symptoms. Patients with very low CoQ10 (<390 microg/L) suffered significantly more from concentration and memory disturbances.



The results show that lowered levels of CoQ10 play a role in the pathophysiology of ME/CFS and that symptoms, such as fatigue, and autonomic and neurocognitive symptoms may be caused by CoQ10 depletion. Our results suggest that patients with ME/CFS would benefit from CoQ10 supplementation in order to normalize the low CoQ10 syndrome and the IO&NS disorders. The findings that lower CoQ10 is an independent predictor of chronic heart failure (CHF) and mortality due to CHF may explain previous reports that the mean age of ME/CFS patients dying from CHF is 25 years younger than the age of those dying from CHF in the general population. Since statins significantly decrease plasma CoQ10, ME/CFS should be regarded as a relative contraindication for treatment with statins without CoQ10 supplementation.

title: Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)
abstract: There is evidence that myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by activation of immune, inflammatory, oxidative and nitrosative stress (IO&NS) pathways. The present study was carried out in order to examine whether ME/CFS is accompanied by increased levels of plasma peroxides and serum oxidized LDL (oxLDL) antibodies, two biomarkers of oxidative stress.  Blood was collected from 56 patients with ME/CFS and 37 normal volunteers. Severity of ME/CFS was measured using the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale.  Plasma peroxide concentrations were significantly higher in patients with ME/CFS than in normal controls. There was a trend towards significantly higher serum oxLDL antibodies in ME/CFS than in controls. Both biomarkers contributed significantly in discriminating between patients with ME/CFS and normal controls. Plasma peroxide and serum oxLDL antibody levels were both significantly related to one of the FF symptoms.  The results show that ME/CFS is characterized by increased oxidative stress.

title: Relationship between musculoskeletal symptoms and blood markers of oxidative stress in patients with chronic fatigue syndrome
abstract: In 21 patients with chronic fatigue syndrome (CFS) versus 20 normal subjects, we investigated the oxidant/antioxidant balance and its correlation with muscle symptoms. Patients versus controls showed significantly: lower Lag Phase and Vitamin E (Vit E) concentrations in plasma and low-density lipoproteins (LDL), higher LDL thiobarbituric acid reactive substances (TBARS), higher fatigue and lower muscle pain thresholds to electrical stimulation. A significant direct linear correlation was found between fatigue and TBARS, thresholds and Lag Phase, thresholds and Vit E in plasma and LDL. A significant inverse linear correlation was found between fatigue and Lag Phase, fatigue and Vit E, thresholds and TBARS. Increased oxidative stress and decreased antioxidant defenses are related to the extent of symptomatology in CFS, suggesting that antioxidant supplementation might relieve muscle symptoms in the syndrome.

title: Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms.
abstract: The aetiology of chronic fatigue syndrome (CFS) is unknown; however, recent evidence suggests excessive free radical (FR) generation may be involved. This study investigated for the first time levels of 8-iso-prostaglandin-F(2 alpha)-isoprostanes alongside other plasma markers of oxidative stress in CFS patients and control subjects. Forty-seven patients (18 males, 29 females, mean age 48 [19--63] years) who fulfilled the Centres for Disease Control classification for CFS and 34 healthy volunteers (13 males, 21 females, 46 [19--63] years) were enrolled in the study. The CFS patients were divided into two groups; one group had previously defined cardiovascular (CV) risk factors of obesity and hypertension (group 1) and the second were normotensive and nonobese (group 2). Patients had significantly increased levels of isoprostanes (group 1, P=0.007; group 2, P=0.03, unpaired t test compared to controls) and oxidised low-density lipoproteins (group 2, P=0.02) indicative of a FR attack on lipids. CFS patients also had significantly lower high-density lipoproteins (group 1, P=0.011; group 2, P=0.005). CFS symptoms correlated with isoprostane levels, but only in group 2 low CV risk CFS patients (isoprostanes correlated with; total symptom score P=0.005; joint pain P=0.002; postexertional malaise P=0.027, Pearson). This is the first time that raised levels of the gold standard measure of in vivo oxidative stress (isoprostanes) and their association with CFS symptoms have been reported.

title: Blood parameters indicative of oxidative stress are associated with symptom expression in chronic fatigue syndrome
abstract: Full blood counts, ESR, CRP, haematinics and markers for oxidative stress were measured for 33 patients diagnosed with chronic fatigue syndrome (CFS) and 27 age and sex matched controls. All participants also completed symptom questionnaires. CFS patients had increases in malondialdehyde (P <0.006), methaemoglobin (P <0.02), mean erythrocyte volume (P <0.02) and 2,3-diphosphoglycerate (P <0.04) compared with controls. Multiple regression analysis found methaemoglobin to be the principal component that differentiated between CFS patients and control subjects. Methaemoglobin was found to be the major component associated with variation in symptom expression in CFS patients (R2 = 0.99, P <0.00001), which included fatigue, musculoskeletal symptoms, pain and sleep disturbance. Variation in levels of malondialdehyde and 2,3-diphosphoglycerate were associated with variations in cognitive symptoms and sleep disturbance (R2 = 0.99, P <0.00001). These data suggest that oxidative stress due to excess free radical formation is a contributor to the pathology of CFS and was associated with symptom presentation.

title: Increased ventricular lactate in chronic fatigue syndrome. III. Relationships to cortical glutathione and clinical symptoms implicate oxidative stress in disorder pathophysiology
abstract: Chronic fatigue syndrome (CFS) is a complex illness, which is often misdiagnosed as a psychiatric illness. In two previous reports, using 1H MRSI, we found significantly higher levels of ventricular cerebrospinal fluid (CSF) lactate in patients with CFS relative to those with generalized anxiety disorder and healthy volunteers (HV), but not relative to those with major depressive disorder (MDD). In this third independent cross‐sectional neuroimaging study, we investigated a pathophysiological model which postulated that elevations of CSF lactate in patients with CFS might be caused by increased oxidative stress, cerebral hypoperfusion and/or secondary mitochondrial dysfunction. Fifteen patients with CFS, 15 with MDD and 13 HVs were studied using the following modalities: (i) 1H MRSI to measure CSF lactate; (ii) single‐voxel 1H MRS to measure levels of cortical glutathione (GSH) as a marker of antioxidant capacity; (iii) arterial spin labeling (ASL) MRI to measure regional cerebral blood flow (rCBF); and (iv) 31P MRSI to measure brain high‐energy phosphates as objective indices of mitochondrial dysfunction. We found elevated ventricular lactate and decreased GSH in patients with CFS and MDD relative to HVs. GSH did not differ significantly between the two patient groups. In addition, we found lower rCBF in the left anterior cingulate cortex and the right lingual gyrus in patients with CFS relative to HVs, but rCBF did not differ between those with CFS and MDD. We found no differences between the three groups in terms of any high‐energy phosphate metabolites. In exploratory correlation analyses, we found that levels of ventricular lactate and cortical GSH were inversely correlated, and significantly associated with several key indices of physical health and disability. Collectively, the results of this third independent study support a pathophysiological model of CFS in which increased oxidative stress may play a key role in CFS etiopathophysiology.

title: Fluctuation of serum vitamin E (α-tocopherol) concentrations during exacerbation and remission phases in patients with chronic fatigue syndrome
abstract: The etiology of chronic fatigue syndrome remains unknown. Oxidative stress may be involved in its pathogenesis. Vitamin E is a major endogenous lipid-soluble antioxidative substance, and is consumed during the lipid peroxidation process. We studied a population comprising 27 patients with chronic fatigue syndrome (10 men and 17 women, 29 ± 6 years of age) and 27 age- and sex-matched control subjects. Serum vitamin E (α-tocopherol) concentrations were determined and expressed as mg/g total lipids (total cholesterol and triglyceride) to evaluate oxidative stress. Serum α-tocopherol concentrations (mg/g lipids) were significantly (P < 0.001) lower in the patients with chronic fatigue syndrome (2.81 ± 0.73) than in the control subjects (3.88 ± 0.65). The patients with chronic fatigue syndrome were re-examined during a follow-up interval. After 8 ± 2 months, 16 patients exhibited a status that warranted re-examination during remission of the symptoms at a regular visit to our hospital (Group 1), while the remaining 11 did not (Group 2). The serum α-tocopherol levels were significantly elevated during remission as compared with those at baseline in Group 1 (2.71 ± 0.62 → 3.24 ± 0.83, P < 0.001). The levels did not significantly change after the interval in Group 2 (2.97 ± 0.86 → 2.85 ± 0.73, not significant). In conclusion, serum α-tocopherol concentrations were significantly lower in the patients with chronic fatigue syndrome as compared with the control subjects, suggesting increased oxidative stress in the former. The low level of serum α-tocopherol was ameliorated during the remission phase as compared with the exacerbation phase in the patients with chronic fatigue syndrome, suggesting that increased oxidative stress may be involved in the pathogenesis of chronic fatigue syndrome and might also be directly related to the severity of the symptoms of chronic fatigue syndrome.

title: Magnesium Status and Parameters of the Oxidant-Antioxidant Balance in Patients with Chronic Fatigue: Effects of Supplementation with Magnesium
abstract: Magnesium deficiency and oxidative stress have both been identified as pathogenic factors in aging and in several age-related diseases. The link between these two factors is unclear in humans although, in experimental animals, severe Mg deficiency has been shown to lead to increased oxidative stress.  The relationship between Mg body stores, dietary intakes and supplements on the one hand and parameters of the oxidant-antioxidant balance on the other was investigated in human subjects.  The study population consisted of 93 patients with unexplained chronic fatigue (median age 38 years, 25% male, 16% smokers and 54% with Chronic Fatigue Syndrome (CFS). Mg deficient patients (47%) had lower total antioxidant capacity in plasma (p=0.007) which was related to serum albumin. Mg deficient patients whose Mg body stores did not improve after oral supplementation with Mg (10 mg/kg/day) had persistently lower blood glutathione levels (p=0.003). In vitro production of thiobarbituric acid reactive substances (TBARS) by non-HDL lipoproteins incubated with copper was related to serum cholesterol (p<0.001) but not to Mg or antioxidants and did not improve after Mg supplementation. In contrast, velocity of formation of fluorescent products of peroxidation (slope) correlated with serum vitamin E (p<0.001), which was, in turn, related to Mg dietary intakes. Both slope and serum vitamin E improved after Mg supplementation (p<0.001).  These results show that the lower antioxidant capacity found in moderate Mg deficiency was not due to a deficit in Mg dietary intakes and was not accompanied by increased lipid susceptibility to in vitro peroxidation. Nevertheless, Mg supplementation was followed by an improvement in Mg body stores, in serum vitamin E and its interrelated stage of lipid peroxidation.

title: Antioxidant status and lipoprotein peroxidation in chronic fatigue syndrome.
abstract: The aetiology and pathogenesis of the Chronic Fatigue Syndrome (CFS) are still largely unresolved. Accompanying metabolic disorders such as selective n-6 fatty acid depletion suggest that oxidative stress and more specifically lipid peroxidation might play a role in its pathogenesis. In order to investigate this hypothesis, oxidant-antioxidant status and its impact on lipoprotein peroxidation in vitro was examined in 61 patients with unexplained fatigue lasting more than 1 month. They were subdivided into 2 groups: group CFS+ (33 subjects) fulfilled the 1988 Center of Disease Control criteria for CFS and group CFS- did not but was similar as regards age, sex distribution and clinical characteristics. Antioxidant status was similar in the 2 groups except for lower serum transferrin in the CFS + (mean (95 % CI) 2.41 (2.28-2.54) versus 2.73 (2.54-2.92) g/L in the CFS-, p = 0.009) and higher lipoprotein peroxidation in vitro: 6630 (5949-7312) versus 5581 (4852-6310) nmol MDA/mg LDL and VLDL cholesterol x minutes, p = 0.035). CFS intensified the influence of LDL cholesterol (p = 0.012) and of transferrin (p = 0.045) on peroxidation in vitro, suggesting additional pro-oxidant effects. These results indicate that patients with CFS have increased susceptibility of LDL and VLDL to copper-induced peroxidation and that this is related both to their lower levels of serum transferrin and to other unidentified pro-oxidising effects of CFS.

title: Not in the mind but in the cell: increased production of cyclo-oxygenase-2 and inducible NO synthase in chronic fatigue syndrome.
abstract: Chronic fatigue syndrome (CFS) is a medically unexplained disorder, characterized by profound fatigue, infectious, rheumatological and neuropsychiatric symptoms. There is, however, some evidence that CFS is accompanied by signs of increased oxidative stress and inflammation in the peripheral blood. This paper examines the role of the inducible enzymes cyclo-oxygenase (COX-2) and inducible NO synthase (iNOS) in the pathophysiology of CFS. Toward this end we examined the production of COX-2 and iNOS by peripheral blood lymphocytes (PBMC) in 18 CFS patients and 18 normal volunteers and examined the relationships between those inflammatory markers and the severity of illness as measured by means of the FibroFatigue scale and the production of the transcription factor nuclear factor kappa beta (NFkappabeta). We found that the production of COX-2 and iNOS was significantly higher in CFS patients than in normal controls. There were significant and positive intercorrelations between COX-2, iNOS and NFkappabeta and between COX-2 and iNOS, on the one hand, and the severity of illness, on the other. The production of COX-2 and iNOS by PBMCs was significantly related to aches and pain, muscular tension, fatigue, concentration difficulties, failing memory, sadness and a subjective experience of infection. The results suggest that a) an intracellular inflammatory response in the white blood cells plays an important role in the pathophysiology of CFS; b) the inflammatory response in CFS is driven by the transcription factor NFkappabeta; c) symptoms, such as fatigue, pain, cognitive defects and the subjective feeling of infection, indicates the presence of a genuine inflammatory response in CFS patients; and d) CFS patients may be treated with substances that inhibit the production of COX-2 and iNOS.

title: Specific correlations between muscle oxidative stress and chronic fatigue syndrome: a working hypothesis
abstract: Chronic fatigue syndrome (CFS) is a relatively common disorder defined as a status of severe persistent disabling fatigue and subjective unwellness. While the biological basis of the pathology of this disease has recently been confirmed, its pathophysiology remains to be elucidated. Moreover, since the causes of CFS have not been identified, treatment programs are directed at symptom relief, with the ultimate goal of the patient regaining some level of pre-existing function and well-being. Several studies have examined whether CFS is associated with: (i) a range of infectious agents and or immune disturbance; (ii) specific changes of activity in the central or peripheral nervous systems; and (iii) elevated stress periods, which may be associated with the pathology via genetic mechanisms. The role of oxidative stress in CFS is an emerging focus of research due to evidence of its association with some pathological features of this syndrome. New data collectively support the presence of specific critical points in the muscle that are affected by free radicals and in view of these considerations, the possible role of skeletal muscle oxidative imbalance in the genesis of CFS is discussed.

title: Chronic fatigue syndrome and mitochondrial dysfunction.
abstract: This study aims to improve the health of patients suffering from chronic fatigue syndrome (CFS) by interventions based on the biochemistry of the illness, specifically the function of mitochondria in producing ATP (adenosine triphosphate), the energy currency for all body functions, and recycling ADP (adenosine diphosphate) to replenish the ATP supply as needed. Patients attending a private medical practice specializing in CFS were diagnosed using the Centers for Disease Control criteria. In consultation with each patient, an integer on the Bell Ability Scale was assigned, and a blood sample was taken for the "ATP profile" test, designed for CFS and other fatigue conditions. Each test produced 5 numerical factors which describe the availability of ATP in neutrophils, the fraction complexed with magnesium, the efficiency of oxidative phosphorylation, and the transfer efficiencies of ADP into the mitochondria and ATP into the cytosol where the energy is used. With the consent of each of 71 patients and 53 normal, healthy controls the 5 factors have been collated and compared with the Bell Ability Scale. The individual numerical factors show that patients have different combinations of biochemical lesions. When the factors are combined, a remarkable correlation is observed between the degree of mitochondrial dysfunction and the severity of illness (P<0.001). Only 1 of the 71 patients overlaps the normal region. The "ATP profile" test is a powerful diagnostic tool and can differentiate patients who have fatigue and other symptoms as a result of energy wastage by stress and psychological factors from those who have insufficient energy due to cellular respiration dysfunction. The individual factors indicate which remedial actions, in the form of dietary supplements, drugs and detoxification, are most likely to be of benefit, and what further tests should be carried out.

title: Mitochondrial dysfunction and the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).
abstract: The objectives of this study are to test the hypothesis that the fatigue and accompanying symptoms of Chronic Myalgic Encephalomyelitis/Fatigue Syndrome are in part due to defects in energy provision at the cellular level, and to understand the pathophysiology of the defects so that effective medical intervention can be implemented. We performed an audit of 138 patients (ages 18-65) diagnosed with ME/CFS and attending a private practice. The patients and 53 normal, healthy controls had the ATP Profile test carried out on neutrophils from a 3-ml venous blood sample. This test yields 6 numerical factors that describe the availability of ATP and the efficiency of oxidative phosphorylation in mitochondria. Other biomedical measurements, including the concentration of cell-free DNA in plasma, were made. The results of the audit are compared with the controls and a previous cohort of 61 patients. We find that all patients tested have measureable mitochondrial dysfunction which correlates with the severity of the illness. The patients divide into two main groups differentiated by how cellular metabolism attempts to compensate for the dysfunction. Comparisons with exercise studies suggest that the dysfunction in neutrophils also occurs in other cells. This is confirmed by the cell-free DNA measurements which indicate levels of tissue damage up to 3.5 times the normal reference range. The major immediate causes of the dysfunction are lack of essential substrates and partial blocking of the translocator protein sites in mitochondria. The ATP Profile is a valuable diagnostic tool for the clinical management of ME/CFS.

title: In vitro Study of Muscle Aerobic Metabolism in Chronic Fatigue Syndrome
abstract: AbstractThe purpose of this study was to establish if muscle aerobic metabolism is abnormal in chronic fatigue syndrome (CFS). Myoblast cultures from muscle biopsies of 16 patients with CFS and 10 healthy controls were established. Micromethods were used to determine the lactate/pyruvate (L/P) ratio, respiratory chain function and cytochrome oxidase and lactic dehydrogenase activities. Mitochondrial DNA (mtDNA) volume was measured and mtDNA rearrangements sought. The results showed that myoblasts from ten of 16 cases of CFS had defects in aerobic metabolism: two had increased L/P ratios, suggestive of a defect in oxidative phosphorylation while eight had decreased ratios, consistent with a deficiency in pyruvate dehydrogenase. There was a statistically significant broader range of L/P ratios in the patients' cultures, compared to controls (p = 0.011). No mtDNA rearrangements were present. This in vitro study confirms that there is convincing evidence of mild aerobic defects in skeletal muscle in some case...

title: Mitochondrial abnormalities in the postviral fatigue syndrome
abstract: SummaryWe have examined the muscle biopsies of 50 patients who had postviral fatigue syndrome (PFS) for from 1 to 17 years. We found mild to severe atrophy of type II fibres in 39 biopsies, with a mild to moderate excess of lipid. On ultrastructural examination, 35 of these specimens showed branching and fusion of mitochondrial cristae. Mitochondrial degeneration was obvious in 40 of the biopsies with swelling, vacuolation, myelin figures and secondary lysosomes. These abnormalities were in obvious contrast to control biopsies, where even mild changes were rarely detected. The findings described here provide the first evidence that PFS may be due to a mitochondrial disorder precipitated by a virus infection.

title: Abnormalities in pH handling by peripheral muscle and potential regulation by the autonomic nervous system in chronic fatigue syndrome
abstract: Jones DEJ, Hollingsworth KG, Taylor R, Blamire AM, Newton JL (From the Institute of Cellular Medicine, Newcastle Magnetic Resonance Centre, and Institute for Ageing and Health, Newcastle University, UK). Abnormalities in pH handling by peripheral muscle and potential regulation by the autonomic nervous system in chronic fatigue syndrome.  2010;

title: Impaired cardiovascular response to standing in Chronic Fatigue Syndrome
abstract: (7): 608–615

title: Impaired oxygen delivery to muscle in chronic fatigue syndrome.
abstract: The purpose of this study was to determine if chronic fatigue syndrome (CFS) is associated with reduced oxygen delivery to muscles. Patients with CFS according to CDC (Center for Disease Control) criteria (n=20) were compared with normal sedentary subjects (n=12). Muscle oxygen delivery was measured as the rate of post-exercise and post-ischaemia oxygen-haem resaturation. Oxygen-haem resaturation was measured in the medial gastrocnemius muscle using continuous-wavelength near-IR spectroscopy. Phosphocreatine resynthesis was measured simultaneously using (31)P magnetic resonance spectroscopy. The time constant of oxygen delivery was significantly reduced in CFS patients after exercise (46.5+/-16 s; mean+/-S.D.) compared with that in controls (29.4+/-6.9 s). The time constant of oxygen delivery was also reduced (20.0+/-12 s) compared with controls (12.0+/-2.8 s) after cuff ischaemia. Oxidative metabolism was also reduced by 20% in CFS patients, and a significant correlation was found between oxidative metabolism and recovery of oxygen delivery. In conclusion, oxygen delivery was reduced in CFS patients compared with that in sedentary controls. This result is consistent with previous studies showing abnormal autonomic control of blood flow. Reduced oxidative delivery in CFS patients could be specifically related to CFS, or could be a non-specific effect of reduced activity levels in these patients. While these results suggest that reduced oxygen delivery could result in reduced oxidative metabolism and muscle fatigue, further studies will be needed to address this issue.

title: Reduced oxidative muscle metabolism in chronic fatigue syndrome
abstract: The purpose of this study was to determine if chronic fatigue syndrome (CSF) is characterized by abnormalities in oxidative muscle metabolism. Patients with CFS according to Centers for Disease Control (CDC) criteria (n = 22) were compared to normal sedentary subjects (n = 15). CFS patients were also tested before and 2 days after a maximal treadmill test. Muscle oxidative capacity was measured as the maximal rate of postexercise phosphocreatine (PCr) resynthesis using the ADP model (Vmax) in the calf muscles using 31P magnetic resonance spectroscopy. Vmax was significantly reduced in CFS patients (39.6 ± 2.8 mmol/L/min, mean ± SE) compared to controls (53.8 ± 2.8 mmol/L/min). Two days postexercise there was no change in resting inorganic phosphate (Pi)/PCr or Vmax in the CFS patients (n = 14). In conclusion, oxidative metabolism is reduced in CFS patients compared to sedentary controls. In addition, a single bout of strenuous exercise did not cause a further reduction in oxidative metabolism, or alter resting Pi/PCr ratios.

title: Skeletal muscle metabolism in the chronic fatigue syndrome. In vivo assessment by 31P nuclear magnetic resonance spectroscopy.
abstract: Previous study of patients with chronic fatigue syndrome (CFS) has demonstrated a markedly reduced dynamic exercise capacity, not limited by cardiac performance and in the absence of clinical neuromuscular dysfunction, suggesting the possibility of a subclinical defect of skeletal muscle.



The in vivo metabolism of the gastrocnemius muscles of 22 CFS patients and 21 normal control subjects was compared during rest, graded dynamic exercise to exhaustion and recovery, using 31P nuclear magnetic resonance (NMR) spectroscopy to reflect minute-to-minute intracellular high-energy phosphate metabolism.



Duration of exercise was markedly shorter in the CFS patients (8.1 +/- 2.8 min) compared with the normal subjects (11.3 +/- 4.3 min) (p = 0.005). There were large changes in phosphocreatine (PCr), inorganic phosphate (Pi), and pH from rest to clinical fatigue in all subjects, reflecting the high intensity of the exercise. The temporal metabolic patterns were qualitatively similar in the CFS patients and normal subjects. There were early and continuous changes in PCr and Pi that peaked at the point of fatigue and rapidly reversed after exercise. In contrast, pH was relatively static in early exercise, not declining noticeably until 50 percent of total exercise duration was achieved, and reaching a nadir at 2 min postexercise, before rapidly reversing. There were no differences in pH at rest (7.08 +/- 0.04 vs 7.10 +/- 0.04), exhaustion (6.85 +/- 0.17 vs 6.76 +/- 0.17) or early (6.64 +/- 0.25 vs 6.56 +/- 0.24) or late recovery (7.09 +/- 0.04 vs 7.10 +/- 0.05), CFS patients vs normal subjects, respectively (NS). Neither were there intergroup differences (NS) in PCr or Pi. Although, quantitatively, the changes in PCr, Pi, and pH were marked and similar in both groups from rest to exhaustion, the changes all occurred much more rapidly in the CFS patients. Moreover, adenosine triphosphate (ATP) was significantly (p = 0.007) less at exhaustion in the CFS group.



Patients with CFS and normal control subjects have similar skeletal muscle metabolic patterns during dynamic exercise and reach similar clinical and metabolic end points. However, CFS patients reach exhaustion much more rapidly than normal subjects, at which point they also have relatively reduced intracellular concentrations of ATP. These data suggest a defect of oxidative metabolism with a resultant acceleration of glycolysis in the working skeletal muscles of CFS patients. This metabolic defect may contribute to the reduced physical endurance of CFS patients. Its etiology is unknown. Whether CFS patients' overwhelming tiredness at rest has a similar metabolic pathophysiology or etiology also remains unknown.

title: EXCESSIVE INTRACELLULAR ACIDOSIS OF SKELETAL MUSCLE ON EXERCISE IN A PATIENT WITH A POST-VIRAL EXHAUSTION/FATIGUE SYNDROME A 31P Nuclear Magnetic Resonance Study
abstract: A patient with prolonged post-viral exhaustion and excessive fatigue was examined by 31P nuclear magnetic resonance. During exercise, muscles of the forearm demonstrated abnormally early intracellular acidosis for the exercise performed. This was out of proportion to the associated changes in high-energy phosphates. This may represent excessive lactic acid formation resulting from a disorder of metabolic regulation. The metabolic abnormality in this patient could not have been demonstrated by traditional diagnostic techniques.

title: Muscle fibre characteristics and lactate responses to exercise in chronic fatigue syndrome
abstract: To examine the proportions of type 1 and type 2 muscle fibres and the degree of muscle fibre atrophy and hypertrophy in patients with chronic fatigue syndrome in relation to lactate responses to exercise, and to determine to what extent any abnormalities found might be due to inactivity.  Quadriceps needle muscle biopsies were obtained from 105 patients with chronic fatigue syndrome and the proportions of type 1 and 2 fibres and fibre atrophy and hypertrophy factors were determined from histochemical preparations, using a semiautomated image analysis system. Forty one randomly selected biopsies were also examined by electron microscopy. Lactate responses to exercise were measured in the subanaerobic threshold exercise test (SATET).  Inactivity would be expected to result in a shift to type 2 fibre predominance and fibre atrophy, but type 1 predominance (23%) was more common than type 2 predominance (3%), and fibre atrophy was found in only 10.4% of cases. Patients with increased lactate responses to exercise did have significantly fewer type 1 muscle fibres (p<0.043 males, p<0.0003 females), but there was no evidence that this group was less active than the patients with normal lactate responses. No significant ultrastructural abnormalities were found.  Muscle histometry in patients with chronic fatigue syndrome generally did not show the changes expected as a result of inactivity. However, patients with abnormal lactate responses to exercise had a significantly lower proportion of mitochondria rich type 1 muscle fibres.

title: Heterogeneity in chronic fatigue syndrome: evidence from magnetic resonance spectroscopy of muscle
abstract: It has been shown previously that some patients with chronic fatigue syndrome show an abnormal increase in plasma lactate following a short period of moderate exercise, in the sub-anaerobic threshold exercise test (SATET). This cannot be explained satisfactorily by the effects of 'inactivity' or 'deconditioning', and patients with abnormal lactate responses to exercise (SATET +ve) have been found to have significantly fewer Type 1 muscle fibres in quadriceps biopsies than SATET -ve patients. We performed phosphorus magnetic resonance spectroscopy on forearm muscles of 10 SATET +ve patients, 9 SATET -ve patients and 13 sedentary volunteers. There were no differences in resting spectra between these groups but at the end of exercise, intracellular pH in the SATET +ve patients was significantly lower than in both the SATET -ve cases and controls (P < 0.03), and the SATET +ve patients also showed a significantly lower ATP synthesis rate during recovery (P < 0.01), indicating impaired mitochondrial oxidative phosphorylation. These observations support other evidence which indicates that chronic fatigue syndrome is a heterogeneous disorder, and confirms the view that some chronic fatigue syndrome patients have a peripheral component to their fatigue.

title: Skeletal muscle bioenergetics in the chronic fatigue syndrome.
abstract: Skeletal muscle bioenergetics and control of intracellular pH have been investigated in 46 patients with chronic fatigue syndrome by phosphorus magnetic resonance spectroscopy. The results have been compared with those from healthy controls and from a group of patients with mitochondrial cytopathies affecting skeletal muscle. No consistent abnormalities of glycolysis, mitochondrial metabolism or pH regulation were identified in the group when taken as a whole, although in 12 of the 46 patients the relationship between pH and phosphocreatine utilisation during exercise fell outside the normal range. Of these, 6 patients showed increased acidification relative to phosphocreatine depletion while 6 showed reduced acidification. These findings do not support the hypothesis that any specific metabolic abnormality underlies fatigue in this syndrome although abnormalities may be present in a minority of patients.

title: Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity
abstract: BackgroundThe aim of this study was to investigate the possibility that a decreased mitochondrial ATP synthesis causes muscular and mental fatigue and plays a role in the pathophysiology of the chronic fatigue syndrome (CFS/ME).MethodsFemale patients (n = 15) and controls (n = 15) performed a cardiopulmonary exercise test (CPET) by cycling at a continuously increased work rate till maximal exertion. The CPET was repeated 24 h later. Before the tests, blood was taken for the isolation of peripheral blood mononuclear cells (PBMC), which were processed in a special way to preserve their oxidative phosphorylation, which was tested later in the presence of ADP and phosphate in permeabilized cells with glutamate, malate and malonate plus or minus the complex I inhibitor rotenone, and succinate with rotenone plus or minus the complex II inhibitor malonate in order to measure the ATP production via Complex I and II, respectively. Plasma CK was determined as a surrogate measure of a decreased oxidative phosphorylation in muscle, since the previous finding that in a group of patients with external ophthalmoplegia the oxygen consumption by isolated muscle mitochondria correlated negatively with plasma creatine kinase, 24 h after exercise.ResultsAt both exercise tests the patients reached the anaerobic threshold and the maximal exercise at a much lower oxygen consumption than the controls and this worsened in the second test. This implies an increase of lactate, the product of anaerobic glycolysis, and a decrease of the mitochondrial ATP production in the patients. In the past this was also found in patients with defects in the mitochondrial oxidative phosphorylation. However the oxidative phosphorylation in PBMC was similar in CFS/ME patients and controls. The plasma creatine kinase levels before and 24 h after exercise were low in patients and controls, suggesting normality of the muscular mitochondrial oxidative phosphorylation.ConclusionThe decrease in mitochondrial ATP synthesis in the CFS/ME patients is not caused by a defect in the enzyme complexes catalyzing oxidative phosphorylation, but in another factor.

title: Decreased oxygen extraction during cardiopulmonary exercise test in patients with chronic fatigue syndrome
abstract: BackgroundThe insufficient metabolic adaptation to exercise in Chronic Fatigue Syndrome (CFS) is still being debated and poorly understood.MethodsWe analysed the cardiopulmonary exercise tests of CFS patients, idiopathic chronic fatigue (CFI) patients and healthy visitors. Continuous non-invasive measurement of the cardiac output by Nexfin® (BMEYE B.V. Amsterdam, the Netherlands) was added to the cardiopulmonary exercise tests. The peak oxygen extraction by muscle cells and the increase of cardiac output relative to the increase of oxygen uptake (ΔQ’/ΔV’O2) were measured, calculated from the cardiac output and the oxygen uptake during incremental exercise.ResultsThe peak oxygen extraction by muscle cells was 10.83 ± 2.80 ml/100ml in 178 CFS women, 11.62 ± 2.90 ml/100 ml in 172 CFI, and 13.45 ± 2.72 ml/100 ml in 11 healthy women (ANOVA: P=0.001), 13.66 ± 3.31 ml/100 ml in 25 CFS men, 14.63 ± 4.38 ml/100 ml in 51 CFI, and 19.52 ± 6.53 ml/100 ml in 7 healthy men (ANOVA: P=0.008).The ΔQ’/ΔV’O2 was > 6 L/L (normal ΔQ’/ΔV’O2 ≈ 5 L/L) in 70% of the patients and in 22% of the healthy group.ConclusionLow oxygen uptake by muscle cells causes exercise intolerance in a majority of CFS patients, indicating insufficient metabolic adaptation to incremental exercise. The high increase of the cardiac output relative to the increase of oxygen uptake argues against deconditioning as a cause for physical impairment in these patients.

title: Ventricular cerebrospinal fluid lactate is increased in chronic fatigue syndrome compared with generalized anxiety disorder: an in vivo 3.0 T 1H MRS imaging study
abstract: Chronic fatigue syndrome (CFS) is a controversial diagnosis because of the lack of biomarkers for the illness and its symptom overlap with neuropsychiatric, infectious, and rheumatological disorders. We compared lateral ventricular volumes derived from tissue‐segmented T1‐weighted volumetric MRI data and cerebrospinal fluid (CSF) lactate concentrations measured by proton MRS imaging (1H MRSI) in 16 subjects with CFS (modified US Centers for Disease Control and Prevention criteria) with those in 14 patients with generalized anxiety disorder (GAD) and in 15 healthy volunteers, matched group‐wise for age, sex, body mass index, handedness, and IQ. Mean lateral ventricular lactate concentrations measured by 1H MRSI in CFS were increased by 297% compared with those in GAD (P < 0.001) and by 348% compared with those in healthy volunteers (P < 0.001), even after controlling for ventricular volume, which did not differ significantly between the groups. Regression analysis revealed that diagnosis accounted for 43% of the variance in ventricular lactate. CFS is associated with significantly raised concentrations of ventricular lactate, potentially consistent with recent evidence of decreased cortical blood flow, secondary mitochondrial dysfunction, and/or oxidative stress abnormalities in the disorder.

title: Mitochondrial dysfunction in bipolar disorder: evidence from magnetic resonance spectroscopy research
abstract: Magnetic resonance spectroscopy (MRS) affords a noninvasive window on in vivo brain chemistry and, as such, provides a unique opportunity to gain insight into the biochemical pathology of bipolar disorder. Studies utilizing proton (1H) MRS have identified changes in cerebral concentrations of N-acetyl aspartate, glutamate/glutamine, choline-containing compounds, myo-inositol, and lactate in bipolar subjects compared to normal controls, while studies using phosphorus (31P) MRS have examined additional alterations in levels of phosphocreatine, phosphomonoesters, and intracellular pH. We hypothesize that the majority of MRS findings in bipolar subjects can be fit into a more cohesive bioenergetic and neurochemical model of bipolar illness that is both novel and yet in concordance with findings from complementary methodological approaches. In this review, we propose a hypothesis of mitochondrial dysfunction in bipolar disorder that involves impaired oxidative phosphorylation, a resultant shift toward glycolytic energy production, a decrease in total energy production and/or substrate availability, and altered phospholipid metabolism.

title: Mitochondrial dysfunction in schizophrenia: Pathways, mechanisms and implications
abstract: Mitochondria play a critical role in regulating cellular functions including bioenergetics, calcium homeostasis, redox signalling, and apoptotic cell death. Mitochondria are also essential to many aspects of neurodevelopment and neuronal functions. However, mitochondrial impairment may affect bioenergetics in the developing brain and alter critical neuronal processes leading to neurodevelopmental abnormalities. Schizophrenia is a chronic and severe neuropsychiatric disorder of neurodevelopmental origin. Immuno-inflammatory pathway is one of the widely appreciated mechanisms that has consistently been implicated in the neurodevelopmental origin of schizophrenia. However, the source of inflammation and the underlying neurobiological mechanisms leading to schizophrenia are yet to be fully ascertained. Recent understanding reveals that perturbation of mitochondrial network dynamics might lead to various nervous system disorders with inflammatory pathologies. Mitochondrial deficit, altered redox balance and chronic low-grade inflammation are evident in schizophrenia. It is hypothesized that oxidative/nitrosative stress responses due to mitochondrial dysfunctions might activate immuno-inflammatory pathways and subsequently lead to neuroprogressive changes in schizophrenia. Herein, we summarise the current understanding of molecular links between mitochondrial dysfunctions and pathogenesis of schizophrenia based on evidence from genomics, proteomics and imaging studies, which together support a role for mitochondrial impairment in the pathogenetic pathways of schizophrenia.

title: Autoimmune diseases, bipolar disorder, and non-affective psychosis.
abstract: Clinic-based studies of immune function, as well as comorbidity of autoimmune diseases, bipolar disorder, and schizophrenia, suggest a possible autoimmune etiology. Studies of non-affective psychosis and schizophrenia suggest common etiologies. The objective was to determine the degree to which 30 different autoimmune diseases are antecedent risk factors for bipolar disorder, schizophrenia, and non-affective psychosis.



A cohort of 3.57 million births in Denmark was linked to the Psychiatric Case Register and the National Hospital Register. There were 20,317 cases of schizophrenia, 39,076 cases of non-affective psychosis, and 9,920 cases of bipolar disorder.



As in prior studies, there was a range of autoimmune diseases which predicted raised risk of schizophrenia in individuals who had a history of autoimmune diseases, and also raised risk in persons whose first-degree relatives had an onset of autoimmune disease prior to onset of schizophrenia in the case. These relationships also existed for the broader category of non-affective psychosis. Only pernicious anemia in the family was associated with raised risk for bipolar disorder (relative risk: 1.7), suggesting a small role for genetic linkage. A history of Guillain-Barré syndrome, Crohn's disease, and autoimmune hepatitis in the individual was associated with raised risk of bipolar disorder.



The familial relationship of schizophrenia to a range of autoimmune diseases extends to non-affective psychosis, but not to bipolar disorder. The data suggest that autoimmune processes precede onset of schizophrenia, but also non-affective psychosis and bipolar disorder.

title: Serological observations in patients suffering from acute manic episodes
abstract: Although abnormalities of the immune system have been described in depression, information on serological alteration in acutely manic patients has been scarce. The present study undertook to investigate the levels of C‐reactive proteins, circulating immune complexes, total immunoglobulins and immunoglobulin subclasses, complement proteins C3, C4, C6 and Factor B in the sera of 45 patients suffering from an acute manic episode. The findings were compared with assessments on the sera of 45 controls. The results demonstrate a number of significant differences between patients and controls. Whilst levels of immunoglobulin D were significantly lower, the levels of total immunoglobulin and immunoglobulin G1, complement proteins C3, C6 and Factor B were raised in the patient group when compared with the controls. Our results suggest a relationship between acute mania and immunological parameters associated with acute phase responses.

title: Activation of indices of cell-mediated immunity in bipolar mania
abstract: Evidence supports that macrophages as well as lymphocytes and their products may be involved in the pathophysiology of psychiatric disorders. Whether patients with bipolar disorder have activation or reduction of immunity during a manic episode remains unclear.



The purpose of this case-control study was to investigate the lymphocyte proliferation to phytohemagglutinin (PHA), concanavalin A, and pokeweed mitogen, and plasma levels of soluble interleukin-2 receptor (sIL-2R) and sIL-6R in patients with bipolar mania (DSM-III-R). The subjects were 23 physically healthy patients with Young Mania Rating Scale (YMRS) scores > or = 26 as well as aged < or = 45 years and 23 age- and gender-matched normal control subjects. The above immune variables were measured in acute mania and consequent remission (YMRS scores < or = 12) among bipolar patients.



The lymphocyte proliferation to PHA and the plasma sIL-2R levels, but not sIL-6R, of bipolar patients were significantly higher in acute mania than in consequent remission. These elevations were not due to differences in medication status. Only in acute mania were the plasma sIL-2R levels of patients significantly higher than control subjects. A positive correlation between the changes of manic severity and plasma sIL-2R levels was observed. Remitted bipolar patients and normal control subjects did not differ in any of these measures.



Cell-mediated immunity activation in bipolar mania was demonstrated and may be through a specifically state-dependent immune response.

title: 3-Nitrotyrosine and glutathione antioxidant system in patients in the early and late stages of bipolar disorder.
abstract: There has been an increasing interest in the role of oxidative stress in the pathophysiology of bipolar disorder. To explore this further, we evaluated the activity of glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), as well as 3-nitrotyrosine levels and carbonyl content in patients in the early (within 3 years of illness onset) and late (a minimum of 10 years of illness) stages of bipolar disorder.



We matched 30 patients in the early stage and 30 patients in the late stage of bipolar disorder, diagnosed according to DSM-IV criteria, with 60 healthy controls (30 matched for each group of patients). We measured symptomatic status using the Hamilton Rating Scale for Depression and the Young Mania Rating Scale.



We found a significant increase in 3-nitrotyrosine levels among patients in the early (p < 0.010) and late (p < 0.010) stages of bipolar disorder. The activity of GR and GST was increased only among patients in the late stage of illness. Glutathione peroxidase activity and carbonyl content did not differ among the groups.



Limitations of our study include its cross-sectional design, which did not allow us to examine direct causative mechanisms or the effects of progression of illness, and the potential environmental bias introduced by comparing patient groups recruited from different regions of the world.



Our data indicate a possible tyrosine nitration-induced damage in patients with bipolar disorder that is present from the early stage of illness. Our data also indicate that patients in the late stage of illness demonstrate enhanced activity of GR and GST, which could suggest the involvement of a compensatory system in bipolar disorder.

title: Cytokines in bipolar disorder vs. healthy control subjects: a systematic review and meta-analysis.
abstract: Bipolar disorder may be associated with peripheral immune system dysfunction; however, results in individual studies are conflicting. Our aim was to systematically review evidence of peripheral cytokine alterations in bipolar disorder integrating findings from various affective states.



We conducted a meta-analysis of studies comparing peripheral cytokine concentrations in patients with bipolar disorder with healthy control subjects. Results were reported according to the PRISMA statement.



Eighteen studies with a total of 761 bipolar disorder patients and 919 healthy controls were included. Overall, concentrations of soluble Interleukin (IL)-2 receptor (sIL-2R), tumor necrosis factor-α (TNF-α), soluble tumor necrosis factor receptor type 1 (sTNFR1) (p < 0.001 each), sIL-6R (p = 0.01) and IL-4 (p = 0.04) were significantly higher in bipolar patients compared with healthy controls. There were no significant differences between bipolar disorder patients and healthy control subjects for IL-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IL-1β, IL-1 receptor antagonist (IL-1RA), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1) and sTNFR2.



Employing a global approach, incorporating evidence across affective states, this meta-analysis found some support for peripheral inflammatory alterations in bipolar disorder. Results were limited by heterogeneity between studies, insufficient standardization and lacking control for confounders in individual studies. Further research exploring the role of the peripheral inflammatory system in relation to neuroinflammation is warranted.

title: Inflamed moods: A review of the interactions between inflammation and mood disorders
abstract: Mood disorders have been recognized by the World Health Organization (WHO) as the leading cause of disability worldwide. Notwithstanding the established efficacy of conventional mood agents, many treated individuals continue to remain treatment refractory and/or exhibit clinically significant residual symptoms, cognitive dysfunction, and psychosocial impairment. Therefore, a priority research and clinical agenda is to identify pathophysiological mechanisms subserving mood disorders to improve therapeutic efficacy. During the past decade, inflammation has been revisited as an important etiologic factor of mood disorders. Therefore, the purpose of this synthetic review is threefold: 1) to review the evidence for an association between inflammation and mood disorders, 2) to discuss potential pathophysiologic mechanisms that may explain this association and 3) to present novel therapeutic options currently being investigated that target the inflammatory-mood pathway. Accumulating evidence implicates inflammation as a critical mediator in the pathophysiology of mood disorders. Indeed, elevated levels of pro-inflammatory cytokines have been repeatedly demonstrated in both major depressive disorder (MDD) and bipolar disorder (BD) patients. Further, the induction of a pro-inflammatory state in healthy or medically ill subjects induces 'sickness behavior' resembling depressive symptomatology. Potential mechanisms involved include, but are not limited to, direct effects of pro-inflammatory cytokines on monoamine levels, dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, pathologic microglial cell activation, impaired neuroplasticity and structural and functional brain changes. Anti-inflammatory agents, such as acetyl-salicylic acid (ASA), celecoxib, anti-TNF-α agents, minocycline, curcumin and omega-3 fatty acids, are being investigated for use in mood disorders. Current evidence shows improved outcomes in mood disorder patients when anti-inflammatory agents are used as an adjunct to conventional therapy; however, further research is needed to establish the therapeutic benefit and appropriate dosage.

title: Oxidative stress in psychiatric disorders: evidence base and therapeutic implications.
abstract: Oxidative stress has been implicated in the pathogenesis of diverse disease states, and may be a common pathogenic mechanism underlying many major psychiatric disorders, as the brain has comparatively greater vulnerability to oxidative damage. This review aims to examine the current evidence for the role of oxidative stress in psychiatric disorders, and its academic and clinical implications. A literature search was conducted using the Medline, Pubmed, PsycINFO, CINAHL PLUS, BIOSIS Preview, and Cochrane databases, with a time-frame extending to September 2007. The broadest data for oxidative stress mechanisms have been derived from studies conducted in schizophrenia, where evidence is available from different areas of oxidative research, including oxidative marker assays, psychopharmacology studies, and clinical trials of antioxidants. For bipolar disorder and depression, a solid foundation for oxidative stress hypotheses has been provided by biochemical, genetic, pharmacological, preclinical therapeutic studies and one clinical trial. Oxidative pathophysiology in anxiety disorders is strongly supported by animal models, and also by human biochemical data. Pilot studies have suggested efficacy of N-acetylcysteine in cocaine dependence, while early evidence is accumulating for oxidative mechanisms in autism and attention deficit hyperactivity disorder. In conclusion, multi-dimensional data support the role of oxidative stress in diverse psychiatric disorders. These data not only suggest that oxidative mechanisms may form unifying common pathogenic pathways in psychiatric disorders, but also introduce new targets for the development of therapeutic interventions.

title: Decreased levels of glutathione, the major brain antioxidant, in post-mortem prefrontal cortex from patients with psychiatric disorders.
abstract: Accruing data suggest that oxidative stress may be a factor underlying the pathophysiology of bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ). Glutathione (GSH) is the major free radical scavenger in the brain. Diminished GSH levels elevate cellular vulnerability towards oxidative stress; characterized by accumulating reactive oxygen species. The aim of this study was to determine if mood disorders and SCZ are associated with abnormal GSH and its functionally related enzymes. Post-mortem prefrontal cortex from patients with BD, MDD, SCZ, and from non-psychiatric comparison controls were provided by the Stanley Foundation Neuropathology Consortium. Spectrophotometric analysis was utilized for the quantitative determination of GSH, while immunoblotting analyses were used to examine expression of glutamyl-cysteine ligase (GCL), GSH reductase (GR), and GSH peroxidase (GPx). We found that the levels of reduced, oxidized, and total GSH were significantly decreased in all psychiatric conditions compared to the control group. Although GCL and GR levels did not differ between groups, the levels of GPx were reduced in MDD and SCZ compared to control subjects. Since oxidative damage has been demonstrated in MDD, BD, and SCZ, our finding that GSH levels are reduced in post-mortem prefrontal cortex suggests that these patient groups may be more susceptible to oxidative stress.

title: An updated meta-analysis of oxidative stress markers in bipolar disorder
abstract: Despite its debilitating symptoms, the pathophysiology of bipolar disorder (BD) remains unclear. One consistently compelling finding, however, has been the presence of oxidative stress. In the present investigation, we conducted a meta-analysis of studies that measured oxidative stress markers in BD patients compared to healthy controls. Search terms and selection criteria were determined a priori to identify and include all studies that measured a marker of oxidative stress in BD compared to healthy controls. Eight markers were included: superoxide dismutase, catalase, protein carbonyl, glutathione peroxidase, 3-nitrotyrosine, lipid peroxidation, nitric oxide, and DNA/RNA damage. A meta-analysis of standardized means was conducted using a random-effects model with generic inverse weighting. Between-study heterogeneity, publication bias, and sensitivity analyses were also examined for each marker. Twenty-seven papers were included in the meta-analysis, which comprised a total of 971 unique patients with BD and 886 healthy controls. Lipid peroxidation, DNA/RNA damage, and nitric oxide were significantly increased in BD patients compared to healthy controls. Additionally, the effect size for lipid peroxidation was very high. Publication bias was not detected for any of the markers. The main limitations in this meta-analysis are the high degree of heterogeneity between studies and the small number of studies used in the analysis of some markers. Additionally, the sensitivity analysis indicated that some results are not very robust. The results from this meta-analysis support the role of oxidative stress in bipolar disorder, especially to DNA, RNA, and lipids.

title: Oxidative stress markers in bipolar disorder: a meta-analysis.
abstract: Oxidative stress is thought to mediate neuropathological processes of a number of neuropsychiatric disorders and recent data suggest that oxidative stress may be involved in the pathophysiology of bipolar disorder (BD). In the present investigation, we conducted a meta-analysis of studies that evaluated markers of oxidative stress in individuals with BD, as compared to healthy controls.



A Medline search was conducted to identify studies that measured peripheral markers of oxidative stress in bipolar disorder. Data were subjected to meta-analysis using a random effects model to examine the effect sizes of the pooled results. Bias assessment (Egger's test) and assessment of heterogeneity (I(2)) were also carried out.



Thiobarbituric acidic reactive substances (TBARS) (p = 0.001) as well as NO activity (p = 0.02) were significantly increased in BD with a large effect size for TBARS and a moderate effect size for increase in NO. No significant effect sizes were observed for the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase (all p>0.05).



Some caution is warranted in interpreting these results: (1) Egger's test was positive for SOD, suggesting that SOD results may have been influenced by a publication bias. (2) We analyzed the absolute values of each antioxidant enzyme separately and the literature suggests that an imbalance between the antioxidant enzymes is a better indication of the presence of oxidative stress.



The present meta-analysis suggests that oxidative stress markers are increased in BD and that oxidative stress may play a role in the pathophysiology of BD.

title: Oxidative imbalance in bipolar disorder subtypes: A comparative study
abstract: The oxidants are related with the membrane-associated pathologies in the central nervous system and may have an important role in neuropsychiatric disorders. Several studies were performed on the effects of free radicals in bipolar disorder. However, there are no studies investigating the effects of free radicals both in the subtypes of BD (Bipolar disorders I and II) and in antidepressant induced mania (AIM). In this study, we aimed to investigate the status of oxidative metabolism in BD and its subtypes.



94 bipolar patients (BD I-II and AIM) diagnosed according to DSM IV and as control group 41 healthy subjects were included to the study. The total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) were examined in the properly obtained plasma samples of subjects and healthy controls included in the study.



The patients' TAS, TOS and OSI were significantly higher than the controls. TAS is negatively correlated with the number of previous total episodes in BD I. The BD I group appeared to have higher TOS compared to BD II group.



Oxidative balance is impaired in bipolar disorder. Antioxidant levels may be increased compensatorily in response to increased oxidant levels. Another important result of our study was that in the comparison of the three disease subtypes BD I group was found to have higher TOS compared to the BD II group. This finding is compatible with the literature on BD I and may be associated with the more severe course of BD I.

title: Evaluation of antioxidant enzymes activities and lipid peroxidation in schizophrenic patients treated with typical and atypical antipsychotics
abstract: Studies performed in schizophrenia patients have generally suggested the presence of a compromised antioxidant system, but this is not always consistent with specific observed parameters, which on the whole, show evidences of dysregulation. There are also controversies regarding the oxidative stress status in patients treated with typical vs. atypical antipsychotics. In this context, the aim of the present work was to evaluate the specific activity of some peripheral antioxidant defences like superoxide dismutase (SOD) and glutathione peroxidase (GPX) and the level of a lipid peroxidation maker (malondialdehyde-MDA), in schizophrenic patients treated with typical (haloperidol) or atypical (olanzapine, quetiapine and risperidone) antipsychotics, compared with age-matched healthy subjects. We found a significant decrease in GPX specific activity and also a significant increase of MDA levels in schizophrenic patients, compared to age-matched control group, regardless of their type of treatment. Additionally, an increase in SOD specific activity was observed, mainly in the patients treated with haloperidol and quetiapine. Further research is necessary in order to elucidate the effects of different antipsychotic agents on antioxidant enzymes and lipid peroxidation or possible interventions at the oxidative stress level in schizophrenic patients.

title: Lithium and valproate modulate antioxidant enzymes and prevent ouabain-induced oxidative damage in an animal model of mania.
abstract: In this study, we assessed the oxidative stress parameters in rats submitted to an animal model of mania induced by ouabain (OUA), which included the use of lithium (Li) and valproate (VPA). Li and VPA treatment reversed and prevented the OUA-induced damage in these structures, however, this effect varies depending on the brain region and treatment regimen. Moreover, the activity of the antioxidant enzymes, namely, superoxide dismutase (SOD) and catalase (CAT) was found to be increased and decreased, respectively, in the brain of OUA-administered rats. Li and VPA modulated SOD and CAT activities in OUA-subjected rats in both experimental models. Our results support the notion that Li and VPA exert antioxidant-like properties in the brain of rats submitted to animal model of mania induced by ouabain.

title: Role of glutathione in neuroprotective effects of mood stabilizing drugs lithium and valproate
abstract: Mood stabilizing drugs lithium and valproate are the most commonly used treatments for bipolar disorder. Previous studies in our laboratory indicate that chronic treatment with lithium and valproate inhibits oxidative damage in primary cultured rat cerebral cortical cells. Glutathione, as the major antioxidant in the brain, plays a key role in defending against oxidative damage. The purpose of this study was to determine the role of glutathione in the neuroprotective effects of lithium and valproate against oxidative damage. We found that chronic treatment with lithium and valproate inhibited reactive oxygen metabolite H(2)O(2)-induced cell death in primary cultured rat cerebral cortical cells, while buthionine sulfoximine, an inhibitor of glutathione rate-limiting synthesis enzyme glutamate-cysteine ligase, reduced the neuroprotective effect of lithium and valproate against H(2)O(2)-induced cell death. Further, we found that chronic treatment with lithium and valproate increased glutathione levels in primary cultured rat cerebral cortical cells and that the effects of lithium and valproate on glutathione levels were dose-dependent in human neuroblastoma SH-SY5Y cells. Chronic treatment with lithium and valproate also increased the expression of glutamate-cysteine ligase in both rat cerebral cortical cells and SH-SY5Y cells. In addition, chronic treatment with other mood stabilizing drugs lamotrigine and carbamazepine, but not antidepressants desipramine and fluoxetine, increased both glutathione levels and the expression of glutamate-cysteine ligase in SH-SY5Y cells. These results suggest that glutathione plays an important role in the neuroprotective effects of lithium and valproate, and that glutathione may be a common target for mood stabilizing drugs.

title: The course of nitric oxide and superoxide dismutase during treatment of bipolar depressive episode.
abstract: Studies have already pointed out a possible pathophysiological role of oxidative and antioxidative molecules in bipolar disorder. We aimed to evaluate the activity and levels of antioxidant superoxide dismutase (SOD), and oxidant nitric oxide (NO), in bipolar I depressive episode (BD-DE) patients in a prospective design.



30 BD-DE patients, diagnosed according to DSM IV, and 30 healthy volunteer controls were included. The serum levels of NO and SOD have been studied when admitted to hospital (1st) and on the 30th days. Clinical outcome was measured by Hamilton Depression Scale (HAM-D). The patients were allowed to have their treatments. One patient was dropped out due to insufficient sampling.



As in the previous studies, NO 1st day levels were significantly higher in patients and SOD 1st day activity was significantly low (p<0.01). NO levels significantly decreased (p<0.01) and normalized, as SOD activity significantly increased but did not reach to the controls' levels (p<0.01) on the 30th day.



Despite normalized NO levels, persistent low SOD activity might point out an oxidative imbalance in BD-DE. Chronic low SOD activity may be associated with incapacity of coping with oxidative stress. This research connotes the probable oxidative imbalance in BD-DE and discusses that phenomenon within the continuum of the disease state.

title: Oxidative & nitrosative stress in depression: Why so much stress?
abstract: Many studies support a crucial role for oxidative & nitrosative stress (O&NS) in the pathophysiology of unipolar and bipolar depression. These disorders are characterized inter alia by lowered antioxidant defenses, including: lower levels of zinc, coenzyme Q10, vitamin E and glutathione; increased lipid peroxidation; damage to proteins, DNA and mitochondria; secondary autoimmune responses directed against redox modified nitrosylated proteins and oxidative specific epitopes. This review examines and details a model through which a complex series of environmental factors and biological pathways contribute to increased redox signaling and consequently increased O&NS in mood disorders. This multi-step process highlights the potential for future interventions that encompass a diverse range of environmental and molecular targets in the treatment of depression.

title: Cerebral metabolic rates for glucose in mood disorders. Studies with positron emission tomography and fluorodeoxyglucose F 18.
abstract: Cerebral metabolic rates for glucose were examined in patients with unipolar depression (N = 11), bipolar depression (N = 5), mania (N = 5), bipolar mixed states (N = 3), and in normal controls (N = 9) using positron emission tomography and fluorodeoxyglucose F 18. All subjects were studied supine under ambient room conditions with eyes open. Bipolar depressed and mixed patients had supratentorial whole brain glucose metabolic rates that were significantly lower than those of the other comparison groups. The whole brain metabolic rates for patients with bipolar depression increased going from depression or a mixed state to a euthymic or manic state. Patients with unipolar depression showed a significantly lower ratio of the metabolic rate of the caudate nucleus, divided by that of the hemisphere as a whole, when compared with normal controls and patients with bipolar depression.

title: Mitochondrial dysfunction in bipolar disorder.
abstract: Mitochondrial dysfunction is implicated in bipolar disorder based on the following lines of evidence: 1) Abnormal brain energy metabolism measured by 31P-magnetic resonance spectroscopy, that is, decreased intracellular pH, decreased phosphocreatine (PCr), and enhanced response of PCr to photic stimulation. 2) Possible role of maternal inheritance in the transmission of bipolar disorder. 3) Increased levels of the 4977-bp deletion in mitochondrial DNA (mtDNA) in autopsied brains. 4) Comorbidity of affective disorders in certain types of mitochondrial disorders, such as autosomal inherited chronic progressive external ophthalmoplegia and mitochondrial diabetes mellitus with the 3243 mutation. Based on these findings, we searched for mtDNA mutations/ polymorphisms associated with bipolar disorder and found that 5178C and 10398A polymorphisms in mtDNA were risk factors for bipolar disorder. The 5178C genotype was associated with lower brain intracellular pH. mtDNA variations may play a part in the pathophysiology of bipolar disorder through alteration of intracellular calcium signaling systems. The mitochondrial dysfunction hypothesis, which comprehensively accounts for the pathophysiology of bipolar disorder, is proposed.

title: Abnormalities in mitochondrial structure in cells from patients with bipolar disorder.
abstract: Postmortem, genetic, brain imaging, and peripheral cell studies all support decreased mitochondrial activity as a factor in the manifestation of Bipolar Disorder (BD). Because abnormal mitochondrial morphology is often linked to altered energy metabolism, we investigated whether changes in mitochondrial structure were present in brain and peripheral cells of patients with BD. Mitochondria from patients with BD exhibited size and distributional abnormalities compared with psychiatrically-healthy age-matched controls. Specifically, in brain, individual mitochondria profiles had significantly smaller areas, on average, in BD samples (P = 0.03). In peripheral cells, mitochondria in BD samples were concentrated proportionately more within the perinuclear region than in distal processes (P = 0.0008). These mitochondrial changes did not appear to be correlated with exposure to lithium. Also, these abnormalities in brain and peripheral cells were independent of substantial changes in the actin or tubulin cytoskeleton with which mitochondria interact. The observed changes in mitochondrial size and distribution may be linked to energy deficits and, therefore, may have consequences for cell plasticity, resilience, and survival in patients with BD, especially in brain, which has a high-energy requirement. The findings may have implications for diagnosis, if they are specific to BD, and for treatment, if they provide clues as to the underlying pathophysiology of BD.

title: Acute mania is accompanied by elevated glutamate/glutamine levels within the left dorsolateral prefrontal cortex
abstract: RationaleThe dorsolateral prefrontal cortex (DLPFC) participates in the pathophysiology of mania. In particular, left-sided structural and metabolic abnormalities have been described.ObjectivesClinical symptoms may be due to hyperactivity of cortical glutamatergic neurons, resulting in increased excitatory neurotransmitter flux and thus enhanced Glx levels.MethodsGlutamate/glutamine (Glx) levels were assessed by proton magnetic resonance spectroscopy (1H-MRS) in eight acute manic patients compared with age- and gender-matched controls.ResultsManic patients had significantly elevated Glx levels (t-test; t=–3.1, P=0.008) within the left DLPFC.ConclusionsOur results indicate that the prefrontal cortical glutamatergic system is involved in the pathophysiology of acute mania. This may have implications for the treatment of mania.

title: Reduced intracellular pH in the basal ganglia and whole brain measured by 31P‐MRS in bipolar disorder
abstract: The authors have previously reported that intracellular pH measured by phosphorus‐31 magnetic resonance spectroscopy (31P‐MRS) was decreased in the frontal lobes of patients with bipolar disorder. In the present study, phosphorus metabolism in the basal ganglia was examined in 13 patients with bipolar disorder and 10 matched controls by localized 31P‐MRS. While no significant alteration of peak area ratios was found for all phosphorus metabolites, intracellular pH was significantly reduced in the basal ganglia in patients with bipolar disorder (7.014 ± 0.045) compared with control subjects (7.066 ± 0.047, P < 0.05). Unexpectedly, non‐localized 31P‐MR spectra also showed significantly lower levels of intracellular pH (6.970 ± 0.025) than controls (6.986 ± 0.024, P < 0.05). These results suggest that decreased intracellular pH in the brain of patients with bipolar disorder is not caused by dysfunction of the frontal lobes but reflect altered metabolism at the cellular level.

title: Elevated Cerebrospinal Fluid Lactate Concentrations in Patients with Bipolar Disorder and Schizophrenia: Implications for the Mitochondrial Dysfunction Hypothesis
abstract: Evidence is accumulating that mitochondrial dysfunction is involved in the pathophysiology of bipolar disorder and schizophrenia. Cerebrospinal fluid (CSF) concentration of lactate, a product of extra-mitochondrial glucose metabolism, is commonly elevated in individuals with mitochondrial disorders, especially those with neuropsychiatric symptoms. We tested the hypothesis that patients with bipolar disorder and schizophrenia would, on average, have elevated CSF lactate concentrations compared with healthy control subjects.



The CSF lactate and CSF and plasma glucose concentrations were measured with a YSI (YSI, Yellow Springs, Ohio) 2300 STAT Plus Glucose & Lactate Analyzer in 15 samples from each of three groups of subjects: bipolar I disorder patients, schizophrenic patients, and healthy control subjects.



Mean CSF lactate concentrations were significantly higher in bipolar (1.76 +/- .38) and schizophrenic subjects (1.61 +/- .31) compared with control subjects (1.31 +/- .21 mmol/L). These differences persisted after adjusting means for CSF glucose concentration, which correlated positively with CSF lactate concentration.



This is the first report of increased CSF lactate concentrations in patients with bipolar disorder and schizophrenia. Elevated CSF lactate indicates increased extra-mitochondrial and anaerobic glucose metabolism and is consistent with impaired mitochondrial metabolism. Measuring CSF lactate concentration might help identify bipolar and schizophrenic patients with mitochondrial dysfunction who might benefit from research to elucidate and ultimately rectify possible mitochondrial pathology underlying these disorders.

title: Molecular evidence for mitochondrial dysfunction in bipolar disorder.
abstract: The disease mechanism of bipolar disorder remains unknown. Recent studies have provided evidence for abnormal gene expression in bipolar disorder.



To determine the expression of 12558 nuclear genes in the human hippocampus in healthy control subjects and those with bipolar disorder or schizophrenia.



We used gene arrays to study messenger RNA expression. Data were verified with a real-time quantitative polymerase chain reaction assay.



We studied 10 healthy control subjects, 9 subjects with bipolar disorder, and 8 subjects with schizophrenia.



The expression of nuclear messenger RNA coding for mitochondrial proteins was significantly decreased in the hippocampus in subjects with bipolar disorder but not in those with schizophrenia. Subjects with bipolar disorder were characterized by a pronounced and extensive decrease in the expression of genes regulating oxidative phosphorylation and the adenosine triphosphate-dependent process of proteasome degradation.



These findings point toward a widespread dysregulation of mitochondrial energy metabolism and downstream deficits of adenosine triphosphate-dependent processes in bipolar disorder.

title: Downregulation in components of the mitochondrial electron transport chain in the postmortem frontal cortex of subjects with bipolar disorder.
abstract: Many studies indicate a genetic predisposition to bipolar disorder (BD) and suggest that a number of abnormal genes are involved in its development. In this study, we used DNA microarray technology to analyze gene-expression profiles in the postmortem frontal cortex of subjects with BD.



Microarray hybridization was performed using human 19K microarray with universal human reference RNA in each hybridization. The reference cDNA was labelled with Cy3 and experimental cDNA, with Cy5. Glass array slides were cohybridized with equal amounts of mixed reference and experimental cDNA. Selected gene targets were further verified using real-time polymerase chain reaction (PCR).



We found that 831 genes were differentially expressed in subjects with BD, including a number of genes in the mitochondrial electron transport chain (ETC), phosphatidylinositol-signalling system and glycolysis/ gluconeogenesis. Eight genes coding for the components of the mitochondrial ETC were identified along with 15 others related to mitochondrial function. Downregulation of NADH-ubiquinone oxidoreductase 20-kd subunit (ETC complex I), cytochrome c oxidase polypeptide Vic (ETC complex IV) and ATP synthase lipid-binding protein (ETC complex V) were further verified by real-time PCR. We also found that the expression of the NADH-ubiquinone oxidoreductase 20-kd subunit was increased in subjects with BD who were receiving mood-stabilizing treatment with lithium at the time of death, when compared with subjects with BD who were not being treated with lithium.



Because the mitochondrial ETC is a major source for the generation of reactive oxygen species, these findings suggest that oxidative damage may play an important role in the pathophysiology of BD and that neuroprotection against this damage may be involved in the effect of lithium treatment.

title: Design and rationale of a 16-week adjunctive randomized placebo-controlled trial of mitochondrial agents for the treatment of bipolar depression.
abstract: Bipolar disorder places a significant burden on individuals, caregivers and family, and the broader community. Current treatments are believed to be more effective against manic symptoms, leaving a shortfall in recovery during the depressive phase of the illness. The current study draws on recent evidence suggesting that, in addition to increased oxidative load, alterations in mitochondrial function occur in bipolar disorder.



This 16-week study aims to explore the potential benefits of N-acetylcysteine (NAC) alone or in combination (CT) with selected nutraceuticals believed to enhance mitochondrial function. The study includes adults diagnosed with bipolar disorder currently experiencing an episode of depression. Participants are asked to take NAC, CT, or placebo in addition to any usual treatments. A post-discontinuation visit is conducted 4 weeks following the treatment phase.



The primary outcome of the study will be mean change on the Montgomery-Asberg Depression Rating Scale. Secondary outcomes include functioning, substance use, mania ratings, and quality of life. Blood samples will be collected at baseline and week 16 to explore biochemical alterations following treatment.



This study may provide a novel adjunctive treatment for bipolar depression. Analysis of biological samples may assist in understanding the therapeutic benefits and the underlying etiology of bipolar depression.

title: Immune disturbances during major depression: upregulated expression of interleukin-2 receptors.
abstract: We determined the following immune parameters in drug-free, major depressed patients and in age- and sex-matched healthy controls: the number and percentage of interleukin-2 receptor (IL-2R) bearing cells (CD25+, anti-TAC), serum circulating levels of soluble (s)IL-2Rs, the pre- and postdexamethasone phytohemagglutinin (PHA)-induced accumulation of sIL-2Rs in culture supernatant, and the number of T helper (CD4+) and T suppressor (CD8+) cells. In comparison with normal volunteers, patients with major depression had a higher number and percentage of CD25+ cells, higher concentrations of serum circulating sIL-2Rs, higher supernatant sIL-2Rs after stimulation with PHA, and a higher number of CD4+ cells. The CD4+/CD8+ ratio and the number of CD4+ cells were significantly and positively related to the number of cells expressing the CD25+ antigen. These results may indicate that depressed patients display an increased number of T cells in an early phase of activation.

title: Depression is an inflammatory disease, but cell-mediated immune activation is the key component of depression
abstract: The first findings that depression is characterized by cell-mediated immune activation and inflammation were published between 1990-1993 (Maes et al.). Recently, it was reported that--based on meta-analysis results--depression is an inflammatory disorder because the plasma levels of two cytokines are increased, i.e. interleukin-(IL)-6 and tumor necrosis factor-α (TNFα). The same meta-analysis found that plasma IL-2 and interferon-(IFN)γ levels are not altered in depression, suggesting that there is no T cell activation in that illness. The present paper reviews the body of evidence that depression is accompanied by cell-mediated immune activation. The findings include: increased serum levels of the soluble IL-2 receptor (sIL-2R) and the sCD8 molecule; increased numbers and percentages of T cells bearing T cell activation markers, such as CD2+CD25+, CD3+CD25+, and HLA-DR+; increased stimulated production of IFNγ; higher neopterin and sTNFR-1 or sTNFR-2 levels; induction of indoleamine 2,3-dioxygenase (IDO) with lowered levels of plasma tryptophan and increased levels of tryptophan catabolites along the IDO pathway (TRYCATs); and glucocorticoid resistance in immune cells. Interferon-α (IFNα)-based immunotherapy shows that baseline and IFNα-induced activation of T cells, IDO activity and TRYCAT formation are related to the development of IFNα-induced depressive symptoms. Animal models of depression show that a cell-mediated immune response is related to the development of depression-like behavior. Antidepressants and mood stabilizers suppress different aspects of cell-mediated immunity and rather specifically target IFNγ production. This review shows that inflammation and cell-mediated immune activation are key factors in depression.

title: Activation of cell-mediated immunity in depression: Association with inflammation, melancholia, clinical staging and the fatigue and somatic symptom cluster of depression
abstract: Depression is characterized by activation of cell-mediated immunity (CMI), including increased neopterin levels, and increased pro-inflammatory cytokines (PICs), such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα). These PICs may induce depressive, melancholic and chronic fatigue (CF) symptoms.



We examined serum neopterin and plasma PIC levels in depressive subgroups in relation to the depressive subtypes and the melancholic and CF symptoms of depression. Participants were 85 patients with depression and in 26 normal controls. Severity of depression was assessed with the Hamilton Depression Rating Scale (HDRS) and severity of CF with the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale.



Serum neopterin was significantly higher in depressed patients and in particular in those with melancholia. There were positive correlations between serum neopterin, the plasma PICs and the number of previous depressive episodes. Neopterin and TNFα were associated with melancholia, while both PICs were associated with CF. Melancholia-group membership was predicted by the HDRS and neopterin, and CF group membership by age, the FF score and serum TNFα.



Depression and melancholia are accompanied by CMI activation, suggesting that neopterin plays a role in their pathophysiology, e.g. through activation of oxidative and nitrosative stress and apoptosis pathways. The intertwined CMI and inflammatory responses are potentially associated with the onset of depression and with the melancholic and CF symptoms of depression. Exposure to previous depressive episodes may magnify the size of CMI and PIC responses, possibly increasing the likelihood of new depressive episodes. CMI activation and inflammation may contribute to the staging or recurrence of depression.

title: A Meta-Analysis of Cytokines in Major Depression
abstract: Major depression occurs in 4.4% to 20% of the general population. Studies suggest that major depression is accompanied by immune dysregulation and activation of the inflammatory response system (IRS). Our objective was to quantitatively summarize the data on concentrations of specific cytokines in patients diagnosed with a major depressive episode and controls.



We performed a meta-analysis of studies measuring cytokine concentration in patients with major depression, with a database search of the English literature (to August 2009) and a manual search of references.



Twenty-four studies involving unstimulated measurements of cytokines in patients meeting DSM criteria for major depression were included in the meta-analysis; 13 for tumor necrosis factor (TNF)-alpha, 9 for interleukin (IL)-1beta, 16 for IL-6, 5 for IL-4, 5 for IL-2, 4 for IL-8, 6 for IL-10, and 4 for interferon (IFN)-gamma. There were significantly higher concentrations of TNF-alpha (p < .00001), weighted mean difference (WMD) (95% confidence interval) 3.97 pg/mL (2.24 to 5.71), in depressed subjects compared with control subjects (438 depressed/350 nondepressed). Also, IL-6 concentrations were significantly higher (p < .00001) in depressed subjects compared with control subjects (492 depressed/400 nondepressed) with an overall WMD of 1.78 pg/mL (1.23 to 2.33). There were no significant differences among depressed and nondepressed subjects for the other cytokines studied.



This meta-analysis reports significantly higher concentrations of the proinflammatory cytokines TNF-alpha and IL-6 in depressed subjects compared with control subjects. While both positive and negative results have been reported in individual studies, this meta-analytic result strengthens evidence that depression is accompanied by activation of the IRS.

title: Interleukin (IL)-6, tumour necrosis factor alpha (TNF-α) and soluble interleukin-2 receptors (sIL-2R) are elevated in patients with major depressive disorder: a meta-analysis and meta-regression.
abstract: Many studies have explored the association between soluble interleukin-2 receptor (sIL-2R), cytokines and major depressive disorder (MDD). However, the results of these studies were not consistent. The aim of our study is to compare the levels of sIL-2R and cytokines in the blood between MDD patients and controls by a meta-analysis and to identify moderators accounting for potential heterogeneity in the levels of sIL-2R and cytokines in MDD patients versus controls by meta-regression analyses.



A comprehensive literature search was performed to identify studies comparing the levels of sIL-2R and cytokines between MDD patients and controls. We pooled the effect sizes for standardized mean differences (SMD) of the levels of sIL-2R and cytokines. We also performed meta-regression and sensitivity analyses to investigate the roles of age, gender, sample type, ethnic origin and selected studies' quality in explaining potential heterogeneity and differences in results respectively.



Twenty-nine studies were selected for this analysis. The levels of sIL-2R, TNF-α and IL-6 in MDD patients were significantly higher than those of healthy controls (SMD=0.555, p<0.001, SMD=0.567, p=0.010; SMD=0.680, p<0.001). Mean age of all subjects was a significant moderator to explain the high heterogeneity of IL-6. Sensitivity analysis found that European but not non-European subjects have higher levels difference of sIL-2R, TNF-α and IL-1β between MDD patients and controls.



The severity of MDD was not considered.



The blood levels of sIL-2R, TNF-α and IL-6 were significantly higher in MDD patients than controls. Age, samples source and ethnic origins may play a potential role in heterogeneity.

title: Associations of Depression With C-Reactive Protein, IL-1, and IL-6: A Meta-Analysis
abstract: To assess the magnitude and direction of associations of depression with C-reactive protein (CRP), interleukin (IL)-1, and IL-6 in community and clinical samples.  Systematic review of articles published between January 1967 and January 2008 in the PubMed and PsycINFO electronic databases was performed. Effect sizes were calculated as stat d and meta-analyzed, using random-effects models.  Each inflammatory marker was positively associated with depression; CRP, d = 0.15 (95% CI = 0.10, 0.21), p < .001; IL-6, d = 0.25 (95% CI = 0.18, 0.31), p < .001; IL-1, d = 0.35 (95% CI = 0.03, 0.67), p = .03; IL-1ra, d = 0.25 (95% CI = 0.04, 0.46), p = .02. Associations were strongest in clinically depressed patient samples—but were also significant in community-based samples—and when clinical interviews were used. Studies adjusting for body mass index (BMI) had smaller associations, albeit significant. Relationships were inconsistent with respect to age, medication, and sex. Depression was related to CRP and IL-6 among patients with cardiac disease or cancer.  Depression and CRP, IL-1, and IL-6 are positively associated in clinical and community samples and BMI is implicated as a mediating/moderating factor. Continuity in clinic- and community-based samples suggests there is a dose-response relationship between depression and these inflammatory markers, lending strength to the contention that the cardiac (or cancer) risk conferred by depression is not exclusive to patient populations. Available evidence is consistent with three causal pathways: depression to inflammation, inflammation to depression, and bidirectional relationships. ANS = autonomic nervous system; BDI = Beck Depression Inventory; BMI = body mass index; CAD = coronary artery disease; CES-D = Center for Epidemiological Studies-Depression Scale; CI = confidence interval; CNS = central nervous system; CRH = corticotrophin-releasing hormone; CRP = C-reactive protein; DSM = Diagnostic and Statistical Manual of Mental Disorders; HPA = hypothalamic-pituitary-adrenal; IL = interleukin; IL-1ra = interleukin-1 receptor antagonist; LPS = lipopolysaccharide; MI = myocardial infarction; OTC = over-the-counter; PBMC = Peripheral Blood Mononuclear Cells; PHQ-9 = Depression Module of the Patient Health Questionnaire; SE = Standard Error.

title: Might different cytokine trends in depressed patients receiving duloxetine indicate differential biological backgrounds.
abstract: Correlational studies investigating neurohormonal-cytokine modulation by antidepressants suggest, among others, variations in cytokines balances as state markers of different biological subtypes of major depressive disorder (MDD) and response predictors to specific treatments. Objective of the study was to investigate cytokines modulation by duloxetine, a relatively newer SNRI with "clean" dual serotonin/norepinephrine mechanism.



30 MDD patients and 32 healthy controls were assessed using Hamilton Depression Scale (HAM-D) and monitored for levels of IL-1β, IL-2, IL-4, IL-10, IL-12, IFN-γ and TNF-α, at baseline, week 6 and week 12 of duloxetine treatment (60mg/day) and at baseline, respectively.



Early responders (ER: defined at week 6 by reduction >50% of baseline HAM-D score) and early non-responders (ENR) showed opposite trends in cytokine levels during duloxetine treatment: ENR were characterized by baseline Th2 shift compared to controls (lower IL-1β, IFN-γ and TNF-α) with increase in Th1 cytokines levels during treatment (increase of IL-1β, IL-12, IFN-γ, IL-1β/IL-10 and TNF-α/IL-10, decrease of IL-10), achieving clinical response at week 12; ER were characterized by baseline Th2-to-Th1 relative switch compared to ENR (higher IL-1β, IL-1β/IL-10 and TNF-α/IL-10) with reduction in Th1 cytokines levels during treatment (decrease of TNF-α and TNF-α/IL-10), achieving clinical response at week 6.



Small sample size.



In accordance to early clinical response, duloxetine treatment could divide depressed patients into at least 2 subgroups characterized by clinical and laboratory differentiated behavior, suggesting different neurobiological background within depressive syndrome differentially sensitive to different drug components: pro-serotonergic effect and increase in Th1 cytokines in ENR vs. pro-noradrenergic effect and decrease in Th1 cytokines in ER.

title: Electroretinographic assessment in major depressed patients receiving duloxetine: might differences between responders and non-responders indicate a differential biological background?
abstract: Despite intense research efforts, still too little is known about the biological determinants of depression, thus soliciting diverse study approaches. Among others, the electroretinography (ERG) has been proposed even as a putative proxy (retinal) measurement of central dopaminergic activity for Major Depressive Disorder (MDD) both in drug-naïve patients and subjects receiving antidepressant treatments. Nonetheless, current evidences are merely preliminary, essentially considering just older classes of antidepressants, thus requiring confirmation studies even with newer agents as duloxetine.



Twenty MDD subjects and 20 matched controls received duloxetine 60 mg/day for 12 weeks, being monitored both by standard ERG recording and by administration of the Hamilton scales for Depression and Anxiety and the Young Mania Rating Scale at baseline and week 12 (end of the study).



ERG mean rod b-wave amplitude significantly reduced from baseline to week 12 in those depressed subjects achieving final response (p=.024), decreasing from the highest rank values to the ones, substantially unmodified, seen among non-responders and controls.



Small sample size and lack of multiple assessments.



At least some MDD patients responding to duloxetine might exhibit a peculiar ERG pattern, hypothetically indicating a specific biological background. If confirmed by larger-sampled studies, these results might shed further light in the understanding of the biological determinants of different subtypes of depression, ideally showing alternative patterns of response upon different treatment interventions.

title: Imbalance between pro- and anti-inflammatory cytokines, and between Th1 and Th2 cytokines in depressed patients: the effect of electroacupuncture or fluoxetine treatment.
abstract: An increase in inflammatory response and an imbalance between T-helper (Th) 1 and 2 functions have been implicated in major depression. The aims of the present study were to 1) study the relationship between pro- and anti-inflammatory cytokines and between Th1 and Th2 produced cytokines in depressed patients and 2) evaluate and compare the effect of treatments with electroacupuncture (EA) and fluoxetine on these cytokines.



95 outpatients with major depressive disorder were treated for 6 weeks with EA, fluoxetine or placebo. Hamilton Depression Rating Scale (HDRS) and Clinical Global Impression (CGI) were used to assess severity and therapeutic effects. 30 volunteers served as controls. Serum cytokine concentrations were measured by ELISA.



Increased proinflammatory cytokine interleukin (IL)-1beta and decreased anti-inflammatory cytokine IL-10 were found in the depressed patients. By contract, Th1 produced proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were decreased, and Th2 produced cytokine IL-4 was significantly increased in depressed patients. The ratio of IFN/IL-4 was also increased. Both acupuncture and fluoxetine treatments, but not the placebo, reduced IL-1beta concentrations in responders. However, only acupuncture attenuated TNF-alpha concentration and INF-gamma/IL-4 ratio towards the control level.



These results suggest that an imbalance between the pro- and anti-inflammatory cytokines (IL-1 and IL-10), and between Th1 and Th2 cytokines (INF-gamma or TNF-alpha and IL-4) occurred in untreated depressed patients. Both EA and fluoxetine had an anti-inflammatory effect by reducing IL-1beta. EA treatment also restored the balance between Th1 and Th2 systems by increasing TNF-alpha and decreasing IL-4.

title: Depression and sickness behavior are Janus-faced responses to shared inflammatory pathways
abstract: It is of considerable translational importance whether depression is a form or a consequence of sickness behavior. Sickness behavior is a behavioral complex induced by infections and immune trauma and mediated by pro-inflammatory cytokines. It is an adaptive response that enhances recovery by conserving energy to combat acute inflammation. There are considerable phenomenological similarities between sickness behavior and depression, for example, behavioral inhibition, anorexia and weight loss, and melancholic (anhedonia), physio-somatic (fatigue, hyperalgesia, malaise), anxiety and neurocognitive symptoms. In clinical depression, however, a transition occurs to sensitization of immuno-inflammatory pathways, progressive damage by oxidative and nitrosative stress to lipids, proteins, and DNA, and autoimmune responses directed against self-epitopes. The latter mechanisms are the substrate of a neuroprogressive process, whereby multiple depressive episodes cause neural tissue damage and consequent functional and cognitive sequelae. Thus, shared immuno-inflammatory pathways underpin the physiology of sickness behavior and the pathophysiology of clinical depression explaining their partially overlapping phenomenology. Inflammation may provoke a Janus-faced response with a good, acute side, generating protective inflammation through sickness behavior and a bad, chronic side, for example, clinical depression, a lifelong disorder with positive feedback loops between (neuro)inflammation and (neuro)degenerative processes following less well defined triggers.

title: Pathways underlying neuroprogression in bipolar disorder: Focus on inflammation, oxidative stress and neurotrophic factors
abstract: There is now strong evidence of progressive neuropathological processes in bipolar disorder (BD). On this basis, the current understanding of the neurobiology of BD has shifted from an initial focus on monoamines, subsequently including evidence of changes in intracellular second messenger systems and more recently to, incorporating changes in inflammatory cytokines, corticosteroids, neurotrophins, mitochondrial energy generation, oxidative stress and neurogenesis into a more comprehensive model capable of explaining some of the clinical features of BD. These features include progressive shortening of the inter-episode interval with each recurrence, occurring in consort with reduced probability of treatment response as the illness progresses. To this end, emerging data shows that these biomarkers may differ between early and late stages of BD in parallel with stage-related structural and neurocognitive alterations. This understanding facilitates identification of rational therapeutic targets, and the development of novel treatment classes. Additionally, these pathways provide a cogent explanation for the efficacy of seemingly diverse therapies used in BD, that appear to share common effects on oxidative, inflammatory and neurotrophic pathways.

title: Oxidative/nitrosative stress and immuno-inflammatory pathways in depression: treatment implications.
abstract: This paper reviews recent work on the biological underpinnings of clinical depression emphasizing the crucial role of immunoinflammatory and oxidative and nitrosative stress (O&NS) pathways in driving changes in neuronal regulating tryptophan catabolites (TRYCATs). The essence of the association of O&NS pathways with autoimmune responses in depression is via damage to lipid membranes, anchorage molecules and functional proteins that lead to changes in their chemical structures creating new modified epitopes (neoepitopes), which are highly immunogenic. The abovementioned pathways together with decreased antioxidant levels, including zinc, coenzyme Q10, glutathione and vitamin E, and melatonin are intimately involved in different aspects of depression, including mitochondrial functions and the regulation of cAMP / circadian genes, allowing for impacts across different aspects of symptom patterning. Immuno- inflammatory and O&NS processes may additionally cause alterations in blood-brain barrier permeability and neuroprogression, that is tissue damage, including neurodegeneration and apoptosis, and decreased neurogenesis and neuroplasticity. Activation of those interconnected pathways is relevant to the pathophysiology of acute and chronic depression and the progressive course (staging) of clinical depression. This implies that compounds that target these pathways may be useful to treat acute episodes and prevent further progression of the disease. We herein review some promising compounds, such as melatonin, melatonin receptor agonists, allopregnanolone, PDE4 inhibitors, statins, aspirin, sodium benzoate, tryptophan-enriched diets, and antioxidants, including epigallocatechin gallate, curcumin, quercitin, alpha-lipoic acid and resveratrol.

title: Depression's multiple comorbidities explained by (neuro)inflammatory and oxidative & nitrosative stress pathways.
abstract: There is now evidence that depression, as characterized by melancholic symptoms, anxiety, and fatigue and somatic (F&S) symptoms, is the clinical expression of peripheral cell-mediated activation, inflammation and induction of oxidative and nitrosative stress (IO&NS) pathways and of central microglial activation, decreased neurogenesis and increased apoptosis. This review gives an explanation for the multiple "co-morbidities" between depression and a large variety of a) brain disorders related to neurodegeneration, e.g. Alzheimer's, Parkinson's and Huntington's disease, multiple sclerosis and stroke; b) medical disorders, such as cardiovascular disorder, chronic fatigue syndrome, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, irritable bowel syndrome, leaky gut, diabetes type 1 and 2, obesity and the metabolic syndrome, and HIV infection; and c) conditions, such as hemodialysis, interferon-α-based immunotherapy, the postnatal period and psychosocial stressors. The common denominator of all those disorders/conditions is the presence of microglial activation and/or activation of peripheral IO&NS pathways. There is evidence that shared peripheral and / or central IO&NS pathways underpin the pathophysiology of depression and the previously mentioned disorders and that activation of these IO&NS pathways contributes to shared risk. The IO&NS pathways function as a smoke sensor that detect threats in the peripheral and central parts of the body and signal these threats as melancholic, anxiety, and fatigue and somatic (F&S) symptoms. The presence of concomitant depression is strongly associated with a lower quality of life and increased morbidity and mortality in medical disorders. This may be explained since depression contributes to increased (neuro)inflammatory burden and may therefore drive the inflammatory and degenerative progression. It is concluded that the activation of peripheral and / or central IO&NS pathways may explain the co-occurrence of depression with the above disorders. This shows that depression belongs to the spectrum of inflammatory and degenerative disorders.

title: Is Depression an Inflammatory Disorder?
abstract: Studies consistently report that groups of individuals with major depressive disorder (MDD) demonstrate increased levels of a variety of peripheral inflammatory biomarkers when compared with groups of nondepressed individuals. These findings are often interpreted as meaning that MDD, even in medically healthy individuals, may be an inflammatory condition. In this article, we examine evidence for and against this idea by looking more closely into what the actual patterns of inflammatory findings indicate in terms of the relationship between MDD and the immune system. Data are presented in support of the idea that inflammation only contributes to depression in a subset of patients versus the possibility that the depressogenic effect of inflammatory activation is more widespread and varies depending on the degree of vulnerability any given individual evinces in interconnected physiologic systems known to be implicated in the etiology of MDD. Finally, the treatment implications of these various possibilities are discussed.

title: Lipid peroxidation and antioxidant protection in patients during acute depressive episodes and in remission after fluoxetine treatment.
abstract: Increasing numbers of studies indicate that free radicals and their derivatives play a role in some neuropsychiatric disorders, such as depression. The aim of this study was to investigate the activities of antioxidant enzymes, lipid peroxidation and total antioxidant status (TAS) in patients suffering from major depressive disorder (MDD) as compared to healthy controls. Specifically, we wanted to estimate how fluoxetine influences antioxidant defense and lipid peroxidation. Fifty MDD patients and thirty healthy controls participated in the study. Antioxidant enzyme activities and lipid peroxidation levels were measured in erythrocytes, while TAS was measured in plasma. All measurements were taken during an acute depressive episode and then again during depression remission after a three-month fluoxetine treatment. During acute depressive episodes, patients had significantly higher activity levels of antioxidant enzymes, such as copper-zinc superoxide dismutase (SOD1) and catalase (CAT), as compared to healthy controls. Concentrations of malondialdehyde (MDA) were also significantly higher during depressive episodes. Activity levels of glutathione peroxidase (GPx) did not differ significantly between depressed patients and healthy control subjects. Moreover, the plasma total antioxidant status of the depressed patients was decreased in comparison to control subjects. After three months of fluoxetine treatment, the above parameters did not change significantly. Major depressive disorder is accompanied by disturbances in the balance between pro- and anti-oxidative processes; however, these disturbances do not improve in patients in remission after three months of fluoxetine therapy.

title: Increased Serum Levels of 8-Hydroxy-2′-Deoxyguanosine in Clinical Depression
abstract: We sought to understand the pathophysiological effects of depression by examining group differences in serum levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative damage.  Our sample consisted of 169 participants. Eight-four of these participants met diagnostic criteria for clinical depression. The 85 participants in our comparison group were matched on age, gender, and ethnicity to the depressed group. 8-OHdG was measured by enzyme-linked immunosorbent assay.  After adjusting for age, gender, race/ethnicity, years of education, daily smoking, average number of alcoholic drinks per week, average amount of physical activity per week, and body mass index, participants in the depressed group had significantly higher levels of oxidative DNA damage compared with participants in the control group. Pairwise comparisons showed that participants with major depression had significantly higher levels of 8-OHdG than control subjects and marginally higher levels of 8-OHdG compared with those with minor depression. Furthermore, participants with recurrent episodes of depression had more oxidative damage than participants with single episodes, who in turn had more damage than healthy control subjects. Finally, participants with recurrent episodes of major depression had more DNA damage than other depressed participants, who in turn had more damage than healthy control subjects.  Our findings suggest that increased oxidative damage may represent a common pathophysiological mechanism, whereby depressed individuals become vulnerable to comorbid medical illness. 8-OHdG = 8-hydroxy-2′-deoxyguanosine; DSM-IV= Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; DISH = Depression Interview and Structured Hamilton Interview; ELISA = enzyme-linked immunosorbent assay; HPLC-EC = high-performance liquid chromatography with electrochemical detection; GC-MS= gas chromatography–mass spectrometry; BMI= body mass index.

title: Elevated Level of DNA Damage and Impaired Repair of Oxidative DNA Damage in Patients with Recurrent Depressive Disorder
abstract: Depressive disorder (DD), including recurrent DD (rDD), is a severe psychological disease, which affects a large percentage of the world population. Although pathogenesis of the disease is not known, a growing body of evidence shows that inflammation together with oxidative stress may contribute to development of DD. Since reactive oxygen species produced during stress may damage DNA, we wanted to evaluate the extent of DNA damage and efficiency of DNA repair in patients with depression.  We measured and compared the extent of endogenous DNA damage – single- and double-strand breaks, alkali-labile sites, and oxidative damage of the pyrimidines and purines – in peripheral blood mononuclear cells isolated from rDD patients (n=40) and healthy controls (n=46) using comet assay. We also measured DNA damage evoked by hydrogen peroxide and monitored changes in DNA damage during repair incubation.  We found an increased number DNA breaks, alkali-labile sites, and oxidative modification of DNA bases in the patients compared to the controls. Exposure to hydrogen peroxide evoked the same increased damage in both groups. Examination of the repair kinetics of both groups revealed that the lesions were more efficiently repaired in the controls than in the patients.  For the first time we showed that patients with depression, compared with non-depresses individuals, had more DNA breaks, alkali-labile sites, and oxidative DNA damage, and that those lesions may be accumulated by impairments of the DNA repair systems. More studies must be conducted to elucidate the role of DNA damage and repair in depression.

title: Lower serum vitamin E concentrations in major depression. Another marker of lowered antioxidant defenses in that illness.
abstract: Major depression is associated with defective antioxidant defenses. Vitamin E is the major fat soluble antioxidant in the body. The aim of the present study is to examine serum vitamin E concentrations in major depressed patients versus normal volunteers.



Serum vitamin E concentrations were measured in 26 healthy volunteers and 42 major depressed patients by means of HPLC. Since vitamin E is a fat soluble vitamin, and serum vitamin E concentrations are strongly related to these of low-density-lipoprotein cholesterol (LDL-C) and triglycerides, we have adjusted the results for possible differences in these lipids. The numbers of peripheral blood leukocytes were measured.



Patients with major depression had significantly lower serum vitamin E concentrations than healthy controls. The area under the ROC (receiver operating characteristics) curve was 83%. There were significant and negative correlations between serum vitamin E and number of total leukocytes and neutrophils.



Major depression is accompanied by significantly lower serum vitamin E concentrations, suggesting lower antioxidant defenses against lipid peroxidation. The results could, in part, explain previous findings, which suggest increased lipid peroxidation in major depression.

title: Lipid Peroxidation and Depressed Mood in Community-Dwelling Older Men and Women
abstract: It has been hypothesized that cellular damage caused by oxidative stress is associated with late-life depression but epidemiological evidence is limited. In the present study we evaluated the association between urinary 8-iso-prostaglandin F2α (8-iso-PGF2α), a biomarker of lipid peroxidation, and depressed mood in a large sample of community-dwelling older adults. Participants were selected from the Health, Aging and Body Composition study, a community-based longitudinal study of older persons (aged 70–79 years). The present analyses was based on a subsample of 1027 men and 948 women free of mobility disability. Urinary concentration of 8-iso-PGF2α was measured by radioimmunoassay methods and adjusted for urinary creatinine. Depressed mood was defined as a score greater than 5 on the 15-item Geriatric Depression Scale and/or use of antidepressant medications. Depressed mood was present in 3.0% of men and 5.5% of women. Depressed men presented higher urinary concentrations of 8-iso-PGF2α than non-depressed men even after adjustment for multiple sociodemographic, lifestyle and health factors (p = 0.03, Cohen’s d = 0.30). This association was not present in women (depressed status-by-sex interaction p = 0.04). Our study showed that oxidative damage may be linked to depression in older men from a large sample of the general population. Further studies are needed to explore whether the modulation of oxidative stress may break down the link between late-life depression and its deleterious health consequences.

title: Depressive symptoms are independently correlated with lipid peroxidation in a female population: comparison with vitamins and carotenoids.
abstract: Lipid peroxidation (LPO) is involved in oxidative tissue injuries. The present investigation examined the association between LPO and psychological depressive symptoms.



A cross-sectional study was conducted on 66 female volunteers aged 38-70. Lipid peroxides (LOOH) in serum were evaluated by hemoglobin-methylene blue (Hb-MB) method; additionally, serum antioxidants were also detected. To assess depressive symptoms, the Center for Epidemiologic Studies Depression (CES-D) Scale and a subscale in the 28-item General Health Questionnaire (GHQ) were applied.



LOOH concentration displayed a significant positive correlation with CES-D and GHQ depression scores. Multiple regression analysis was performed in which LOOH concentration served as a dependent variable and CES-D scores and antioxidants as independent variables. Consequently, CES-D scores demonstrated significant positive correlation with LOOH.



The positive relationship between depressive symptoms and LPO in a female population may support the hypothesis that LPO may affect depressive symptoms.

title: Depression and oxidative damage to lipids
abstract: Depression is associated with increased morbidity and mortality from cardiovascular and cerebrovascular diseases. Oxidative damage to lipids is one of the key early events in the etiology of atherosclerosis, the pathologic condition that underlies these diseases. The current study examines the pathophysiological consequences of depression by comparing serum levels of F(2α)-isoprostanes (8-iso-PGF(2α)), a biomarker of oxidative damage to lipids, in a group of depressed individuals (n=73) and a matched comparison group (n=72). The depressed group had significantly higher levels of serum 8-iso-PGF(2α), while controlling for age, gender, race, years of education, daily smoking, number of alcoholic drinks per week, average amount of physical activity per week, and body mass index. Analyses using interviewer ratings on the Hamilton Scale revealed that, within the depressed cohort, there was no significant association between the severity of symptoms and levels of 8-iso-PGF(2α), suggesting this is a threshold rather than a dose-response relationship. Results extend on our knowledge of depression and oxidative damage to lipids. In conclusion, oxidative damage to lipid molecules may represent a common pathophysiological mechanism by which depressed individuals become more vulnerable to atherosclerosis and its clinical sequelae.

title: Urinary excretion of biopyrrins, oxidative metabolites of bilirubin, increases in patients with psychiatric disorders
abstract: Several authors have suggested that psychological stress induces the production of reactive oxygen species (ROS). Several studies have supported the idea that bilirubin exerts antioxidative effects in vivo, and it was reported psychological stress provokes bilirubin oxidation in vivo [Yamaguchi T., Shioji I., Sugimoto A., Yamaoka M., 2002. Psychological stress increases bilirubin metabolites in human urine. ]. We investigated whether the concentration of bilirubin oxidative metabolites (biopyrrins) is increased in urine from patients with psychiatric disorders. The concentration of biopyrrins in urine of 25 patients with psychiatric disorders (schizophrenia, 15; depression, 10) was compared with 96 healthy volunteers. The concentrations of biopyrrins, as measured by enzyme-linked immunosorbent assay, were normalized to the urinary concentration of creatinine. The concentration of biopyrrins in patients with psychiatric disorders (schizophrenia and depression) was significantly higher than that of healthy volunteers. In schizophrenia, biopyrrins levels correlated with scores of the Brief Psychiatric Rating Scale (BPRS), and in depression, biopyrrins levels correlated with scores of the Hamilton Depression Rating Scale (HAM-D). These finding suggest that psychotic states are associated with an increase in the oxidative metabolites of bilirubin in human urine.

title: The efficacy of adjunctive N-acetylcysteine in major depressive disorder: a double-blind, randomized, placebo-controlled trial.
abstract: Major depressive disorder (MDD) is one of the most common psychiatric disorders, conferring considerable individual, family, and community burden. To date, treatments for MDD have been derived from the monoamine hypothesis, and there is a paucity of emerging antidepressants, especially with novel mechanisms of action and treatment targets. N-acetylcysteine (NAC) is a redox-active glutathione precursor that decreases inflammatory cytokines, modulates glutamate, promotes neurogenesis, and decreases apoptosis, all of which contribute to the neurobiology of depression.



Participants with a current episode of MDD diagnosed according to DSM-IV-TR criteria (N = 252) were treated with NAC or placebo in addition to treatment as usual for 12 weeks and were followed to 16 weeks. Data were collected between 2007 and 2011.



The omnibus interaction between group and visit for the Montgomery-Asberg Depression Rating Scale (MADRS), the primary outcome measure, was not significant (F₁,₅₂₀.₉ = 1.98, P = .067), and the groups did not separate at week 12 (t₃₆₀.₃ = -1.12, P = .265). However, at week 12, the scores on the Longitudinal Interval Follow-Up Evaluation-Range of Impaired Functioning Tool (LIFE-RIFT) differed from placebo (P = .03). Among participants with a MADRS score ≥ 25, NAC separated from placebo at weeks 6, 8, 12, and 16 (P < .05). Additionally, the rate of change between baseline and week 16 was significant (t₂₂₁.₀₃ = -2.11, P = .036). NAC treatment was superior to placebo at week 16 for secondary readouts of function and clinical impression. Remission and response were greater in the NAC group at week 16, but not at week 12. The NAC group had a greater rate of gastrointestinal and musculoskeletal adverse events.



Being negative at the week 12 end point, and with some positive secondary signals, the study provides only limited support for the role of NAC as a novel adjunctive therapy for MDD. These data implicate the pathways influenced by NAC in depression pathogenesis, principally oxidative and inflammatory stress and glutamate, although definitive confirmation remains necessary.

title: Increased autoimmune responses against auto-epitopes modified by oxidative and nitrosative damage in depression: implications for the pathways to chronic depression and neuroprogression.
abstract: There is evidence that major depression is characterized by oxidative and nitrosative stress (O&NS). The aim of this study is to examine IgM-mediated autoimmune responses against a variety of modified neo-epitopes formed by O&NS damage to self-epitopes in chronic depression.



Serum IgM antibodies directed against conjugated oleic and azelaic acid, malondialdehyde (MDA), phosphatidyl inositol (Pi), and conjugated nitric-oxide (NO) adducts, i.e., NO-tryptophan, NO-tyrosine, NO-arginine, and NO-cysteinyl, were determined in 33 healthy controls and 74 depressed patients subdivided into 28 patients with chronic (duration >2 year) and 46 without chronic depression.



Serum IgM levels against all neoepitopes were significantly higher in depressed patients than in healthy controls. Moreover, the IgM levels were significantly higher, except Pi, in chronically depressed patients than in non-chronically depressed patients.



Depression is characterized by IgM-related autoimmune responses directed against neo-epitopes that are normally hidden from the immune system but that became immunogenic secondary to damage by O&NS. The results show that the generation of neoantigenic determinants that lead to (auto)immune responses is strongly associated with chronic depression.



The damage caused by O&NS to auto-epitopes and the consequent formation of O&NS modified neoantigenic determinants may increase the risk to develop depression and in particular chronic depression through transition to autoimmune reactions. This has implications for understanding the immuno-inflammatory and oxidative-autoimmune pathways that lead to chronic depression and neuroprogression in that illness.

title: The shared role of oxidative stress and inflammation in major depressive disorder and nicotine dependence
abstract: Nicotine dependence is common in people with mood disorders; however the operative pathways are not well understood. This paper reviews the contribution of inflammation and oxidative stress pathways to the co-association of depressive disorder and nicotine dependence, including increased levels of pro-inflammatory cytokines, increased acute phase proteins, decreased levels of antioxidants and increased oxidative stress. These could be some of the potential pathophysiological mechanisms involved in neuroprogression. The shared inflammatory and oxidative stress pathways by which smoking may increase the risk for development of depressive disorders are in part mediated by increased levels of pro-inflammatory cytokines, diverse neurotransmitter systems, activation the hypothalamic-pituitary-adrenal (HPA) axis, microglial activation, increased production of oxidative stress and decreased levels of antioxidants. Depressive disorder and nicotine dependence are additionally linked imbalance between neuroprotective and neurodegenerative metabolites in the kynurenine pathway that contribute to neuroprogression. These pathways provide a mechanistic framework for understanding the interaction between nicotine dependence and depressive disorder.

title: Mitochondrial dysfunction, oxidative stress, and major depressive disorder
abstract: There is controversy about depression being a physical illness, in part because a reproducible, sensitive, and specific biologic marker is not available. However, there is evidence that mitochondrial dysfunction and oxidative stress may be associated with abnormal brain function and mood disorders, such as depression. This paper reviews selected human and animal studies providing evidence that intracellular mitochondrial metabolic dysfunction in specific brain regions is associated with major depressive disorder. This supports the hypothesis that chronic mitochondrial dysfunction in specific tissues may be associated with depression. Evaluation of mitochondrial dysfunction in specific tissues may broaden the perspective of depression beyond theories about neurotransmitters or receptor sites, and may explain the persistent signs and symptoms of depression.

title: A review on the oxidative and nitrosative stress (O&NS) pathways in major depression and their possible contribution to the (neuro)degenerative processes in that illness
abstract: This paper reviews the body of evidence that major depression is accompanied by a decreased antioxidant status and by induction of oxidative and nitrosative (IO&NS) pathways. Major depression is characterized by significantly lower plasma concentrations of a number of key antioxidants, such as vitamin E, zinc and coenzyme Q10, and a lowered total antioxidant status. Lowered antioxidant enzyme activity, e.g. glutathione peroxidase (GPX), is another hallmark of depression. The abovementioned lowered antioxidant capacity may impair protection against reactive oxygen species (ROS), causing damage to fatty acids, proteins and DNA by oxidative and nitrosative stress (O&NS). Increased ROS in depression is demonstrated by increased levels of plasma peroxides and xanthine oxidase. Damage caused by O&NS is shown by increased levels of malondialdehyde (MDA), a by-product of polyunsaturated fatty acid peroxidation and arachidonic acid; and increased 8-hydroxy-2-deoxyguanosine, indicating oxidative DNA damage. There is also evidence in major depression, that O&NS may have changed inactive autoepitopes to neoantigens, which have acquired immunogenicity and serve as triggers to bypass immunological tolerance, causing (auto)immune responses. Thus, depression is accompanied by increased levels of plasma IgG antibodies against oxidized LDL; and increased IgM-mediated immune responses against membrane fatty acids, like phosphatidyl inositol (Pi); oleic, palmitic, and myristic acid; and NO modified amino-acids, e.g. NO-tyrosine, NO-tryptophan and NO-arginine; and NO-albumin. There is a significant association between depression and polymorphisms in O&NS genes, like manganese superoxide dismutase, catalase, and myeloperoxidase. Animal models of depression very consistently show lowered antioxidant defences and activated O&NS pathways in the peripheral blood and the brain. In animal models of depression, antidepressants consistently increase lowered antioxidant levels and normalize the damage caused by O&NS processes. Antioxidants, such as N-acetyl-cysteine, compounds that mimic GPX activity, and zinc exhibit antidepressive effects. This paper reviews the pathways by which lowered antioxidants and O&NS may contribute to depression, and the (neuro)degenerative processes that accompany that illness. It is concluded that aberrations in O&NS pathways are--together with the inflammatory processes--key components of depression. All in all, the results suggest that depression belongs to the spectrum of (neuro)degenerative disorders.

title: So depression is an inflammatory disease, but where does the inflammation come from?
abstract: BackgroundWe now know that depression is associated with a chronic, low-grade inflammatory response and activation of cell-mediated immunity, as well as activation of the compensatory anti-inflammatory reflex system. It is similarly accompanied by increased oxidative and nitrosative stress (O&NS), which contribute to neuroprogression in the disorder. The obvious question this poses is ‘what is the source of this chronic low-grade inflammation?’DiscussionThis review explores the role of inflammation and oxidative and nitrosative stress as possible mediators of known environmental risk factors in depression, and discusses potential implications of these findings. A range of factors appear to increase the risk for the development of depression, and seem to be associated with systemic inflammation; these include psychosocial stressors, poor diet, physical inactivity, obesity, smoking, altered gut permeability, atopy, dental cares, sleep and vitamin D deficiency.SummaryThe identification of known sources of inflammation provides support for inflammation as a mediating pathway to both risk and neuroprogression in depression. Critically, most of these factors are plastic, and potentially amenable to therapeutic and preventative interventions. Most, but not all, of the above mentioned sources of inflammation may play a role in other psychiatric disorders, such as bipolar disorder, schizophrenia, autism and post-traumatic stress disorder.

title: Major depressive disorder is accompanied with oxidative stress: short‐term antidepressant treatment does not alter oxidative–antioxidative systems
abstract: The aim of the present study was to investigate the oxidative–antioxidative systems and effects of different antidepressants on these systems in patients with major depressive disorder (MDD).

title: Psychosocial Factors as a Potential Trigger of Oxidative DNA Damage in Human Leukocytes
abstract: Although numerous studies have been carried out on the stress‐cancer linkage, the results are still inconclusive. One of the useful, but rarely applied, methods to assess this linkage is to examine the relationship between psychosocial stress and cancer‐predisposing genetic alterations simultaneously. We investigated whether various psychosocial factors can be associated with the levels of 8‐hydroxydeoxyguanosine (8‐OH‐dG), a biomarker of cancer‐related oxidative DNA damage, in peripheral blood leukocytes in 362 healthy workers (276 males and 86 females). After adjustments for age, body mass index, cigarette smoking, and alcohol use, female subjects showed positive relationships between the amount of 8‐OH‐dG and the Tension‐Anxiety, Depression‐Rejection, Anger‐Hostility, Fatigue, and Confusion scores of the Profile of Mood States, respectively. The levels of 8‐OH‐dG also increased reliably in the female subjects who had poor stress‐coping behaviors, particularly wishful thinking strategy, in the NIOSH general job stress instrument. There were positive relationships of the 8‐OH‐dG levels to average working hours, a self‐blame coping strategy, and recent loss of a close family member in male subjects. These findings in a nonclinical sample of healthy adults not only provide evidence of a stress‐cancer linkage, but also suggest possible sex differences in the mechanisms of stress‐related cancer initiation.

title: Depression and possible cancer risk due to oxidative DNA damage.
abstract: The potential link between depression and cancer is an important unsolved question. To clarify this, we compared a cancer-related oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OH-dG), in peripheral leukocytes between 30 patients with depression and 60 age- and gender-matched healthy controls, and examined the 8-OH-dG-related factors. The degree of depression was assessed by the scores of the Center for Epidemiologic Studies Depression scale (CES-D) and the Profile of Mood States (POMS). The patients showed significantly higher 8-OH-dG levels than the controls. There was a significant positive correlation between the CES-D scores and the 8-OH-dG levels in depressive, particularly female, patients. Multiple regression analysis indicated that whether the subjects were patients or controls was a significant predictor of the 8-OH-dG levels in male and total subjects, as was the CES-D score or the Depression-Rejection score of the POMS in female subjects. This study suggests that clinical depression is a risk factor for cancer initiation in view of oxidative DNA damage.

title: Subgenual prefrontal cortex abnormalities in mood disorders
abstract: Pathological disturbances of mood may follow a 'bipolar' course, in which normal moods alternate with both depression and mania, or a 'unipolar' course, in which only depression occurs1–3. Both bipolar and unipolar disorders can be heritable illnesses associated with neurochemical, neuroendocrine and autonomic abnormalities. The neurobiological basis for these abnormalities has not been established2,3. Using positron emission tomographic (PET) images of cerebral blood flow and rate of glucose metabolism to measure brain activity, we have now localized an area of abnormally decreased activity in the pre-frontal cortex ventral to the genu of the corpus callosum in both familial bipolar depressives and familial unipolar depressives. This decrement in activity was at least partly explained by a corresponding reduction in cortical volume4, as magnetic resonance imaging (MRI) demonstrated reductions in the mean grey matter volume in the same area of 39 and 48% in the bipolar and unipolar samples, respectively. This region has previously been implicated in the mediation of emotional and autonomic responses to socially significant or provocative stimuli, and in the modulation of the neurotransmitter systems targeted by antidepressant drugs3,5–10.

title: Review of the literature on major mental disorders in adult patients with mitochondrial diseases.
abstract: Mitochondria are intracellular organelles crucial to the production cellular energy. Mitochondrial disease results from a malfunction in this biochemical cascade. These disorders can affect any organ system, producing diverse signs and symptoms, including psychiatric ones. Several authors argue that mitochondrial dysfunction is related to the pathophysiology of bipolar disorder and schizophrenia. Also, the authors retrieved 19 case reports that describe patients with mitochondrial diseases and psychiatric disorders. Most of these patients have psychiatric presentations that preceded the diagnosis of mitochondrial disease. The most common physical findings are fatigue, muscle weakness with or without atrophy, and hearing loss.

title: A mitochondrial bioenergetic basis of depression
abstract: Major depressive disorder (MDD) is an important public health problem affecting 350 million people worldwide. After decades of study, the pathophysiology of MDD remains elusive, resulting in treatments that are only 30–60 % effective. This review summarizes the emerging evidence that implicates impaired mitochondrial bioenergetics as a basis for MDD. It is suggested that impaired mitochondrial bioenergetic function contributes to the pathophysiology of MDD via several potential pathways including: genetics/genomics, inflammation, oxidative stress, and alterations in neuroplasticity. A discussion of mitochondrial bioenergetic pathways that lead to MDD is provided. Evidence is reviewed regarding the mito-toxic or mito-protective impact of various antidepressant medications currently in use. Opportunities for further research on novel therapeutic approaches, including mitochondrial modulators, as stand-alone or adjunct therapy for reducing depression are suggested. In conclusion, while there is substantial evidence linking mitochondrial bioenergetics and MDD, there are currently no clear mitochondrial phenotypes or biomarkers to use as guides in targeting therapies beyond individuals with MDD and known mitochondrial disorders toward the general population of individuals with MDD. Further study is needed to develop these phenotypes and biomarkers, to identify therapeutic targets, and to test therapies aimed at improving mitochondrial function in individuals whose MDD is to some extent symptomatic of impaired mitochondrial bioenergetics.

title: PET measurements of brain glucose metabolism and blood flow in major depressive disorder: a critical review
abstract: To show that PET investigations of brain function in patients with major depression can contribute with valuable pathophysiological knowledge about brain function of these states.

title: Affective disorders, antidepressant drugs and brain metabolism
abstract: There is increasing evidence that affective disorders are associated with dysfunction of neurotransmitter postsynaptic transduction pathways and that chronic treatment with clinically active drugs results in adaptive modification of these pathways. Despite the close dependence of signal transduction on adenosine triphosphate (ATP) availability, the changes in energy metabolism in affective disorders are largely unknown. This question has been indirectly dealt with through functional imaging studies (PET, SPECT, MRS). Despite some inconsistencies, PET and SPECT studies suggest low activity in cortical (especially frontal) regions in depressed patients, both unipolar and bipolar, and normal or increased activity in the manic pole. Preliminary MRS studies indicate some alterations in brain metabolism, with reduced creatine phosphate and ATP levels in the brain of patients with affective disorders. However, the involvement of the energy metabolism in affective disorders is still debated. We propose direct neurochemical investigations on mitochondrial functional parameters of energy transduction, such as the activities of (a) the enzymatic systems of oxidative metabolic cycle (Kreb's cycle); (b) the electron transfer chain; (c) oxidative phosphorylation, and (d) the enzyme activities of ATP-requiring ATPases. These processes should be studied in affective disorders and in animals treated with antidepressant drugs or lithium.

title: Anatomical and functional correlates in major depressive disorder: the contribution of neuroimaging studies.
abstract: Several studies suggested the neural networks modulating aspects of emotional behaviour to be implicated in the pathophysiology of mood disorders. These networks involve the medial prefrontal cortex (MPFC) and closely related areas in the medial and caudolateral orbital cortex (medial prefrontal network), amygdala, hippocampus, and ventromedial parts of the basal ganglia, where alterations in grey matter volume and neurophysiological activity are found in cases with recurrent depressive episodes. Such findings hold major implications for models of the neurocircuits that underlie depression. In particular, evidence from lesion analysis studies suggests that MPFC and related limbic and striato-pallido-thalamic structures organize emotional expression. The aim of this paper is to review the contribution of the most relevant studies with single photon emission tomography (SPECT), positron emission tomography (PET) and magnetic resonance imaging (MRI) to the understanding of pathophysiology of major depressive disorder (MDD), with particular focus on the reversibility of functional correlates with treatment.

title: Alterations of mitochondrial function and correlations with personality traits in selected major depressive disorder patients.
abstract: Increased occurrence of several physical conditions has been reported in patients with depressive disorders. Various physical conditions and depressive disorder have been reported in patients with mitochondrial disorders. The aim of this study was to investigate mitochondrial function in selected depressed patients in search of an aetiological or pathophysiological factor common to both depression and physical symptoms.



Muscle biopsy was performed in 28 patients with a lifetime prevalence of major depressive disorder (MDD), and at least three chronic physical conditions that have been reported to be common in depressive as well as in mitochondrial disorders. Morphologic and biochemical investigations including mitochondrial ATP production rate (MAPR) by the bioluminometric method, spectrophotometric analyses of mitochondrial enzymes, and long-PCR and Southern blot techniques to detect mitochondrial DNA (mtDNA) deletions were performed. The Karolinska Scales of Personality (KSP) with 15 scales assessing vulnerability to psychopathology was filled in by 21 patients.



Decreases of MAPR and enzyme ratios were found in the patients in comparison with controls (P<0.01). Deletions of mtDNA assessed with long-PCR were more frequent in patients than in controls (chi-square test P<0.05). Correlations were found between MAPR and the KSP scales 'Somatic Anxiety', 'Psychasthenia', and 'Suspicion' (P<0.01).



Results cannot be compared with previous studies, and cannot be generalized to all MDD patients. Individually matched controls were not available.



The results suggest that mitochondrial dysfunction is associated with vulnerability to psychopathology in this selected patient group.

title: Mitochondrial respiration in blood platelets of depressive patients.
abstract: Recent evidences include mitochondrial dysfunctions in pathophysiology of mood disorders. We examined association between depressive disorders and mitochondrial respiration using both intact and permeabilized blood platelets. In intact platelets, physiological respiration, maximal capacity of electron transport system and respiratory rate after complex I inhibition were decreased in depressive patients, who reached partial remission, compared to healthy controls. Respiratory rates were unchanged in several respiratory states in permeabilized platelets. Results indicate that changes in respiratory rate in intact platelets can be used as biological marker of depressive disorder. The hypothesis that decreased mitochondrial respiratory rate participate in pathophysiology of depression was supported.

title: Immunological Dysfunction in Schizophrenia: A Systematic Approach
abstract: In the present study, immunological alterations were investigated as one possible factor contributing towards the pathogenesis of schizophrenia. Specifically cellular changes, deviating cytokine production and interfering variables were studied in order to improve our understanding of how these factors interact.  44 acutely ill schizophrenics were compared with matched healthy controls. Cell numbers were determined by flow cytometry and cytokine production by whole blood assay and ELISA. A criss-cross technique was employed for the assessment of interfering serum factors.  Cell counts for leukocytes, lymphocytes, pan T cells, activated T cells and the absolute B cell count of the schizophrenic patients were all within normal limits. The absolute and relative monocyte counts, the number of IL-2 receptor carrying T cells and the relative B cell count were slightly elevated. IL-2 and IFN-γ production were increased while IL-10 production, the sIL-2R and cortisol levels remained unchanged. No interfering serum factors were detected.  The deficient production of TH-1 cytokines in schizophrenia is not due either to a changed number of immunocompetent cells or to a counterregulation of the TH-2 cytokine IL-10. Serum factors in in vitro testing are not responsible for the deficient cytokine production.

title: Cytokine serum levels, autologous mixed lymphocyte reaction and surface marker analysis in never medicated and chronically medicated schizophrenic patients
abstract: A number of immunological parameters were studied in 82 DSM-III-R diagnosed schizophrenic patients (53 first drug-naive and 29 medicated chronic patients) as well as 62 healthy blood donors. The serum levels of interleukin-2 (IL-2), interleukin-1 beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) were measured and correlated with cellular immunity, as assessed by the autologous mixed lymphocyte reaction (AMLR). T lymphocyte subsets were also examined. The above immune parameters were reassessed in a subgroup of 11 first-episode, drug-naive patients 1month after neuroleptic medication. IL-2 serum levels were significantly lower, and IL-1beta and TNF-alpha were significantly higher in schizophrenic patients compared with healthy donors (P<0.001); no significant difference was observed between the two patient groups (medicated and not medicated). Abnormal cytokine serum levels were associated with decreased AMLR responses in vitro. Increased percentages of activated CD4+ and CD16+ natural killer cells, as well as cells expressing ICAM-1 adhesion molecules and IL-2 specific receptors, were detected in the patients. Immunophenotype studies revealed a higher percentage of cells expressing IL-2 receptors in medicated chronic schizophrenic patients compared with drug-naive patients. The abnormal cytokine production in vivo, along with the low AMLR responses in vitro, and the high percentage of activated CD4+ lymphocytes presented in this study suggest alterations in the immune system of schizophrenic patients (medicated or not medicated) consistent with immune activation.

title: Cytokine Changes and Tryptophan Metabolites in Medication-Naïve and Medication-Free Schizophrenic Patients
abstract: Cytokine imbalances especially between T helper type (Th) 1 and Th2 and tryptophan breakdown were reported to be involved in the pathophysiology of schizophrenia. The hyperactive inflammatory response system could induce enhanced tryptophan breakdown. This study aimed to investigate the relationship between cytokine changes, tryptophan breakdown parameter changes and clinical parameters in patients with schizophrenia in comparison with normal controls. In the plasma of schizophrenic patients, Th1-specific interferon-γ was significantly higher (F = 7.485, p = 0.007) and Th2-specific interleukin (IL)-4 was significantly lower (F = 126.327, p < 0.0001). The Th1-related cytokine IL-2 was lower (F = 5.409, p = 0.021) but tumor necrosis factor-α (TNF-α) and Th2-related IL-6 were higher (F = 95.004, p < 0.0001 and F = 408.176, p < 0.0001, respectively) in the plasma of schizophrenic patients. After 6 weeks of treatment, IL-6 and TNF-α were significantly reduced (t = –3.762, p < 0.0001 and z = –2.668, p = 0.008). At the time of admission, plasma tryptophan concentrations were lower (F = 6.339, p = 0.012) in schizophrenic patients and were negatively correlated with the total positive symptoms score (r2 = -0.343, p = 0.004). After 6 weeks of medication, both plasma tryptophan and kynurenine concentrations were increased (t = –2.937, p = 0.005 and t = –3.214, p = 0.002, respectively). The findings of this study indicate a hyperactive pro-inflammatory response inducing a change in tryptophan metabolism that might be related to the development of positive symptoms in schizophrenia.

title: Th1, Th2 and Th3 cytokine alteration in schizophrenia
abstract: Several studies have shown that there is an imbalance between T helper 1 (Th1) cytokines and T helper 2 (Th2) cytokines in patients with schizophrenia. The T helper 3 (Th3) cytokine, transforming growth factor beta-1 (TGF-beta1), has been shown to suppress the production of Th1 cytokines. Therefore it is hypothesized that it may play a role in schizophrenia by suppressing overactive Th1 system.



We recruited 88 schizophrenic patients and 88 matched controls. The basal plasma concentrations of IFN-gamma (Th1), IL-4 (Th2) and TGF-beta1 (Th3) were studied at the time the patients were admitted to the hospital and following 8 weeks of treatment with antipsychotics.



The detection rate of plasma IFN-gamma and basal plasma TGF-beta1 level were significantly higher in schizophrenic patients than in controls whereas detection rate of plasma IL-4 was lower in patients. The ratio of Th1/Th2 cytokines (IFN-gamma/IL-4) was higher in schizophrenic patients. Following the neuroleptic treatment, the IFNgamma and TGF-beta1 levels returned to control values, and IL-4 concentration rose above the control value.



Schizophrenic patients showed higher Th1/Th2 ratio which is attenuated by effective neuroleptic treatment. It is possible that TGF-beta1 plays a role in reducing the activity of Th1 cytokine.

title: Elevated interleukin-2, interleukin-6 and interleukin-8 serum levels in neuroleptic-free schizophrenia: association with psychopathology
abstract: Cytokines have been one of the recent focal points of immunological research in schizophrenia. The present study was to assess the serum levels of some of interleukins in schizophrenia and their relationships with the psychopathological parameters. Seventy physically healthy Chinese patients, who met DSM-III-R criteria for schizophrenia and who were drug-free for at least 2 weeks, were compared with 30 age- and sex-matched Chinese normal controls. The psychopathology of schizophrenia was assessed by the Positive and Negative Syndrome Scale (PANSS). Serum levels of IL-6 and IL-8 were measured by sandwich enzyme-linked immunosorbent assay (ELISA), and serum IL-2 level was assayed by radioimmunometric assay (RIA). Serum levels of IL-2, IL-6 and IL-8 were significantly elevated in patients with a chronic form of schizophrenia (all p<0.05). There was a significant inverse relationship between IL-2 level and the PANSS positive subscale P (r=-0.31, p=0.006) and a significant positive correlation between IL-8 level and PANSS negative subscale N (r=0.25, p=0.036) in schizophrenic patients. In control subjects, a significant and positive relationship between serum IL-2 and IL-6 (r=0.513, p=0.004) was noted, whereas, there was a significant and negative relationship between IL-2 and IL-8 in schizophrenic patients (r=-0.28, p=0.02). Our data confirms and supports the view that immune disturbance is involved in schizophrenia, which is compatible with the possibility that Chinese schizophrenic patients have an ongoing autoimmune process. This immune disturbance is related to the subgroup of schizophrenic patients with characteristic clinical variables. The dysfunction of interaction or inter-adjustment between different cytokines may exist in schizophrenic patients.

title: Inflammatory Cytokine Alterations in Schizophrenia: A Systematic Quantitative Review
abstract: Cytokines play an important role in infection and inflammation and are crucial mediators of the cross-talk between the brain and the immune system. Schizophrenia would be associated with an imbalance in inflammatory cytokines, leading to a decrease in Th1 and an increase in Th2 cytokine secretion. However, data published so far have been inconsistent. The primary objective of the present meta-analysis was to verify whether the cytokine imbalance hypothesis of schizophrenia is substantiated by evidence.



Cross-sectional studies were included if they assessed in vivo plasma or serum cytokine concentrations and/or in vitro secretion of cytokines by peripheral blood leukocytes from schizophrenia patients and healthy volunteers.



Data from 62 studies involving a total sample size of 2298 schizophrenia patients and 1858 healthy volunteers remained for analysis. Ten cytokines were assessed, including the prototypic Th1 and Th2 cytokines gamma interferon (IFN-gamma) and interleukin 4 (IL-4) as well as IL-2, soluble IL-2 receptor (sIL-2R), IL-1beta, IL-1 receptor antagonist (IL-1RA), tumor necrosis factor-alpha (TNF-alpha), IL-6, soluble IL-6 receptor (sIL-6R), and IL-10. The results show that an increase occurs in in vivo IL-1RA, sIL-2R, and IL-6 and a decrease occurs in in vitro IL-2 in schizophrenia. No significant effect sizes were obtained for the other cytokines.



These findings provide the first evidence of establishment of an inflammatory syndrome in schizophrenia, which refutes the current hypothesis of a Th2 slant. Caveats are presented to data interpretation, including the role of stress and the effect of weight gain that develops in schizophrenia.

title: Th17 pathway-mediated immunopathogenesis of schizophrenia: mechanisms and implications.
abstract: Schizophrenia is a highly complex and severe neuropsychiatric disorder with an unknown etiopathology. Evidence for a dysregulated immune system in both the risk for and progression of schizophrenia has recently been overwhelming. Importantly, chronic low-grade inflammation both in the periphery and central nervous system has been shown to contribute predominantly to the pathogenesis of schizophrenia in a subset of individuals. Inflammation in the central nervous system is mediated by a range of proinflammatory cytokines, resident immune cells such as microglia, and brain infiltrating peripheral immunocompetent cells, such as T lymphocytes. Recently, Th17 cells, a subset of T helper cells have emerged as crucial players in mucosal defense against infections. It is linked to atopic, inflammatory, and autoimmune disorders. The risk factors/mechanisms leading to low-grade inflammation in schizophrenia are diverse and include infectious agents, stress, trauma, environmental toxins, genetic vulnerability, physical inactivity, obesity, poor diet, and sleep disruption. Herein, we propose that fetal programming of cellular immune components driven by intrauterine adversity can lead to the generation of long-lasting effector/memory Th17 cells. Th17 cells can disrupt the blood-brain barrier, infiltrate the central nervous system, and, along with other cytokines and microglia, lead to neuroprogression through neuroinflammation in schizophrenia.

title: The role of pro-inflammatory cytokines in the neuroinflammation and neurogenesis of schizophrenia
abstract: Schizophrenia is a serious mental illness with chronic symptoms and significant impairment in psychosocial functioning. Although novel antipsychotics have been developed, the negative and cognitive symptoms of schizophrenia are still unresponsive to pharmacotherapy. The high level of social impairment and a chronic deteriorating course suggest that schizophrenia likely has neurodegenerative characteristics. Inflammatory markers such as pro-inflammatory cytokines are well-known etiological factors for psychiatric disorders, including schizophrenia. Inflammation in the central nervous system is closely related to neurodegeneration. In addition to pro-inflammatory cytokines, microglia also play an important role in the inflammatory process in the CNS. Uncontrolled activity of pro-inflammatory cytokines and microglia can induce schizophrenia in tandem with genetic vulnerability and glutamatergic neurotransmitters. Several studies have investigated the possible effects of antipsychotics on inflammation and neurogenesis. Additionally, anti-inflammatory adjuvant therapy has been under investigation as a treatment option for schizophrenia. Further studies should consider the confounding effects of systemic factors such as metabolic syndrome and smoking. In addition, the unique mechanisms by which pro-inflammatory cytokines are involved in the etiopathology of schizophrenia should be investigated. In this article, we aimed to review (1) major findings regarding neuroinflammation and pro-inflammatory cytokine alterations in schizophrenia, (2) interactions between neuroinflammation and neurogenesis as possible neural substrates for schizophrenia, and (3) novel pharmacological approaches.

title: Changes in pro-inflammatory cytokines and body weight during 6-month risperidone treatment in drug naïve, first-episode schizophrenia
abstract: present study aimed to examine the changes in pro-inflammatory cytokines and body weight during 6-month risperidone treatment in drug naïve, first-episode schizophrenia.MethodsSixty-two drug naïve, first-episode schizophrenia (SZ group) and 60 healthy individuals (control group) were enrolled in the study. Serum interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) levels, and body weight were measured at baseline for both groups, and repeated for the SZ group at five different time points during 6-month risperidone treatment.ResultsAt baseline, serum IL-1β, IL-6, and TNF-α levels in the SZ group (53.28 ± 12.62, 33.98 ± 14.13, 50.08 ± 12.86 pg/mL, respectively) were significantly higher than those in the control group (23.49 ± 15.27, 15.53 ± 7.16, 32.12 ± 15.23 pg/mL, respectively) (p's < 0.001). Within the SZ group, serum IL-1β levels decreased significantly at 2 weeks (48.02 ± 16.00 pg/mL, p < 0.01) and 1 month (44.70 ± 16.63 pg/mL, p < 0.001), but then gradually increased at 2 months (48.49 ± 18.87 pg/mL), 3 months (50.59 ± 18.48 pg/mL) and 6 months (53.64 ± 16.22 pg/mL) to the levels comparable to baseline; serum IL-6 levels changed significantly over the course of treatment (p = 0.001), but reached the levels comparable to baseline at 6 months (37.13 ± 13.23 pg/mL); serum levels of TNF-α increased significantly at 3 months (55.02 ± 16.69 pg/mL, p < 0.01) and 6 months (58.69 ± 13.57 pg/mL, p < 0.001); steady and significant weight gain was observed at each follow-up time point (p's < 0.001), from 56.71 ± 9.25 kg at baseline to 62.72 ± 9.53 kg at 6 months.ConclusionsRisperidone treatment is associated with changes in serum pro-inflammatory cytokines levels and weight. There is an initial anti-inflammatory effect that reduces with treatment, potentially due to its weight gain side effect.

title: Activation of Th17 cells in drug naïve, first episode schizophrenia
abstract: The present study was to examine the role of pro-inflammatory T helper 17 (Th17) cells in drug naïve, first episode schizophrenia.



Patients with normal weight, drug naïve, first episode schizophrenia and healthy controls were enrolled in the study. Flow cytometric analysis was performed to analyze the proportion of Th17 cells among the CD4(+) T cells. Plasma levels of interleukin-17 (IL-17), interferon-γ (IFN-γ) and interleukin-6 (IL-6) were examined using enzyme-linked immunosorbent assay (ELISA). Psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS). All measures were repeated for the patient group after 4 weeks of risperidone treatment.



Sixty-nine patients with normal weight, drug naïve, first episode schizophrenia and 60 healthy controls were enrolled. At baseline, the patient group hadz significantly higher proportions of Th17 cells and plasma levels of IFN-γ and IL-6 compared with the control group (p's<0.01). Within the patient group, there were significant positive relationships between the proportion of Th17 cells, plasma levels of IL-17, IFN-γ, IL-6 and the PANSS total score after controlling for potential confounding variables (p's<0.05). After 4weeks of risperidone treatment, the proportion of Th17 cells decreased significantly (p <0.001), and there was a significant positive relationship between the PANSS total score change rate and the change in proportion of Th17 cells (p = 0.039).



Patients with normal weight, drug naïve, first episode schizophrenia present activation of Th17 cells, which might be associated with therapeutic response after risperidone treatment.

title: Inflammation and schizophrenia.
abstract: An association between inflammatory abnormalities and schizophrenia has been found repeatedly. The purposes of this special feature are to clarify the key findings on inflammation in schizophrenia, identify major gaps in the literature, and suggest priorities for research in this area.

title: Cognitive impairment is related to oxidative stress and chemokine levels in first psychotic episodes
abstract: This study measures the levels of various markers of oxidative stress and inflammation in blood samples from first-episode psychosis (FEP) patients, and examines the association between these peripheral biomarkers and cognitive performance at 6 months after treatment.



Twenty-eight FEP patients and 28 healthy controls (matched by age, sex and educational level) had blood samples taken at admission for assessment of total antioxidant status, superoxide dismutase (SOD), total glutathione (GSH), catalase (CAT), glutathione peroxidase, lipid peroxidation, nitrites and the chemokine monocyte chemoattractant protein-1 (MCP-1). A battery of cognitive tests was also applied to the healthy controls and those FEP patients who were in remission at 6 months after the acute episode.



FEP patients had significantly lower levels of total antioxidant status, catalase and glutathione peroxidase, compared with the healthy controls. Regression analyses found that MCP-1 levels were negatively associated with learning and memory (verbal and working), nitrite levels were negatively associated with executive function, and glutathione levels were positively associated with executive function.



Our results suggest an association between certain peripheral markers of oxidative stress and inflammation and specific aspects of cognitive functioning in FEP patients. Further studies on the association between MCP-1 and cognition are warranted.

title: Oxidative stress as marker of positive symptoms in schizophrenia
abstract: Schizophrenia, a serious hereditary disease, is a biological disorder of the brain resulting from abnormalities that arise early in life and disrupt a normal development of the brain. The chemical nature of schizophrenic brain is still not completely understood. The brain and nervous system are particularly prone to free radical damage since the membrane lipids are very rich in polyunsaturated fatty acid chains, and areas of human brain are very rich in iron, which plays an essential role in generating free radical species. Following the hypothesis that chronic schizophrenics are under oxidative stress which has exhausted the ability of their antioxidative capacity to adapt the elevated levels of circulating peroxides, we decided to examine the erythrocyte levels of lipid peroxidation products and reduced glutathione and the activities of antioxidative defence enzymes superoxide dismutase (EC 1.15.1.1), glutathione peroxidase (EC 1.11.1.9) and catalase (EC 1.11.1.6) as well as erythrocyte susceptibility to H2O2-induced oxidative stress in schizophrenic patients. The obtained results suggest a misbalance in pro/antioxidant status of chronic schizophrenics, which is more expressed in patients with positive symptoms of the disease.

title: Neurobiology of schizophrenia spectrum disorders: the role of oxidative stress.
abstract: Mitochondrial dysfunction and oxidative stress are increasingly implicated in the pathophysiology of schizophrenia. The brain is the body's highest energy consumer, and the glutathione system is the brain's dominant free radical scavenger. In the current paper, we review the evidence of central and peripheral nervous system anomalies in the oxidative defences of individuals with schizophrenia, principally involving the glutathione system. This is reflected by evidence of the manifold consequences of oxidative stress that include lipid peroxidation, protein carboxylation, DNA damage and apoptosis - all potentially part of the process of neuroprogression in the disorder. Importantly, oxidative stress is amenable to intervention. We consider the clinical potential of some possible interventions that help reduce oxidative stress, via augmentation of the glutathione system, particularly N-acetyl cysteine. We argue that a better understanding of the mechanisms and pathways underlying oxidative stress will assist in developing the therapeutic potential of this area.

title: Oxidative/Nitrosative stress in psychiatric disorders: are we there yet?
abstract: While oxidative stress has a clear role in neurodegenerative diseases, its involvement in psychiatric disorders is only beginning to be understood. Evidence for oxidative stress and redox dysregulation in psychiatric disorders is rapidly mounting, and preclinical data are increasingly suggesting oxidative stress can affect brain circuits involved in schizophrenia, yielding altered behaviors. This issue of Schizophrenia Bulletin covers some recent developments in human and animal studies highlighting the possibility of oxidative and nitrosative stress contributing to abnormal behaviors. Hayes et al 1 present data of inflammation-related measures in patients, at-risk subjects, and healthy controls revealing altered cytokines in the cerebrospinal fluid (CSF) of patients and at-risk mental state subjects. Fournier et al 2 provide an interesting metabolomics approach in cells derived from patients and controls that highlight the possibility of using metabolic signatures of oxidative stress reactivity as biomarkers for prodrome or early psychosis. Overall, the articles point to the use of oxidative/nitrosative stress and inflammation measures as biomarkers for early psychosis and may pave the way to novel therapeutic approaches.

title: Decreased glutathione levels and antioxidant enzyme activities in untreated and treated schizophrenic patients
abstract: There is substantial evidence found in the literature that supports the fact that the presence of oxidative stress may play an important role in the physiopathology of schizophrenia. Previous studies have reported the occurrence of impairments in the glutathione levels and the activities of the antioxidant enzymes in patients suffering from schizophrenia. However, most of these studies were performed on treated patients. The present study evaluated treated schizophrenic patients (n=52) along with neuroleptic-free or untreated schizophrenic patients (n=36) and healthy controls (n=46). The blood glutathione levels: total glutathione (GSHt), reduced glutathione (GSHr), and oxidized glutathione (GSSG) as well as the activities of the antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were measured. The psychopathology of the patients was assessed through the Clinical Global Impressions-severity (CGI-severity). The tests revealed that in comparison with the healthy controls, the schizophrenic patients showed significantly lower levels of GSHr, SOD, and CAT. Among the schizophrenic patients, the activities of the antioxidant enzymes SOD and CAT were recorded to be significantly lower in untreated patients than in the treated ones. In addition, the levels of both GSHt and GSHr were found to be inversely correlated with the obtained CGI-severity score. These results evidently suggest that a decrease in the glutathione levels and the activities of the antioxidant enzymes in patients diagnosed with schizophrenia is not related to neuroleptic treatment and could be considered as a biological indicator of the degree of severity of the symptoms of schizophrenia.

title: Schizophrenia: glutathione deficit in cerebrospinal fluid and prefrontal cortex in vivo
abstract: Schizophrenia is a major psychiatric disease, which affects the centre of the personality, with severe problems of perception, cognition as well as affective and social behaviour. In cerebrospinal fluid of drug‐free schizophrenic patients, a significant decrease in the level of total glutathione (GSH) by 27% (P < 0.05) was observed as compared to controls, in keeping with the reported reduced level of its metabolite γ‐glutamylglutamine. With a new non‐invasive proton magnetic resonance spectroscopy methodology, GSH level in medial prefrontal cortex of schizophrenic patients was found to be 52% (P = 0.0012) lower than in controls. GSH plays a fundamental role in protecting cells from damage by reactive oxygen species generated among others by the metabolism of dopamine. A deficit in GSH would lead to degenerative processes in the surrounding of dopaminergic terminals resulting in loss of connectivity. GSH also potentiates the N‐methyl‐d‐aspartate (NMDA) receptor response to glutamate, an effect presumably reduced by a GSH deficit, leading to a situation similar to the application of phencyclidine (PCP). Thus, a GSH hypothesis might integrate many established biological aspects of schizophrenia.

title: Increased oxidative stress in the anterior cingulate cortex of subjects with bipolar disorder and schizophrenia.
abstract: Recent studies indicate the presence of mitochondrial dysfunction in brains of subjects with bipolar disorder (BD). Because the mitochondrial electron transport chain is a major source for production of reactive oxygen species that cause oxidative stress, we sought to determine in the present study if BD is associated with oxidative stress.



Postmortem anterior cingulate brain sections from subjects with BD, major depressive disorder (MDD), or schizophrenia, and from nonpsychiatric, non-neurologic comparison controls were generously provided by the Stanley Foundation Neuropathology Consortium. Oxidative stress was determined by analyzing 4-hydroxynonenal (4-HNE), a major product of lipid peroxidation. The level of 4-HNE was determined by measuring 4-HNE protein adducts using immunohistochemistry.



We found that 4-HNE levels were significantly increased by 59% in BD subjects and by 47% in schizophrenia subjects, but not in MDD subjects, when compared with controls. Levels of 4-HNE were negatively correlated with pH in all 60 subjects. When pH was used as covariate, 4-HNE levels were still significantly increased in BD subjects when compared with controls. Further, 4-HNE levels were significantly correlated with pH values only in BD subjects, but not in MDD, schizophrenia, or control subjects.



Oxidative damage in the brain may contribute in part to the pathological process in BD and schizophrenia. This finding also suggests antioxidative stress as a probable alternative approach to the pharmacological treatment of these psychiatric disorders.

title: 2-D DIGE analysis of liver and red blood cells provides further evidence for oxidative stress in schizophrenia.
abstract: The molecular disease mechanisms associated with schizophrenia remain largely unknown. Although primarily considered a disorder of the brain, there is evidence of a peripheral component to schizophrenia. In this study, we investigated liver tissue and red blood cells (RBC) from schizophrenia patients and controls using 2-D DIGE proteomic analysis. Fourteen proteins were significantly altered in liver samples from schizophrenia patients (n = 15) compared to healthy controls (n = 15). Analysis of the schizophrenia RBC proteome revealed 8 proteins significantly altered in samples from schizophrenia patients (13 antipsychotic-treated and 7 drug-naïve) compared to controls (n = 20). Six of the altered proteins in the liver and four of the altered RBC proteins are related to oxidative stress. These results corroborate our earlier findings obtained from post-mortem brain studies and substantiate our hypothesis that metabolic alterations leading to oxidative stress are linked to the schizophrenia disease process. Our results also suggest that at least some of the pathological processes associated with the schizophrenia disease process can be traced in peripheral tissue. If peripheral cells can be used as a disease surrogate, promising new investigative avenues could be explored.

title: Reduced status of plasma total antioxidant capacity in schizophrenia
abstract: To examine whether antioxidant capacity is reduced in patients with schizophrenia, we determined plasma total antioxidant status (TAS) by quenching the absorbance of the radical cation formed by the reaction of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) with a metmyoglobin and hydrogen peroxide. TAS serves as an index of net antioxidant activity derived from various antioxidants in plasma. Male schizophrenic patients were compared with age- and sex-matched healthy control subjects, using a within-subject, repeated measures, on-off-on haloperidol treatment design. Drug-free patients were free of all psychotropic medications for an average of 32 days. Plasma TAS was significantly lower in patients with schizophrenia than in normal controls. Plasma TAS in patients was significantly and inversely correlated with symptom severity during the drug-free condition. There were no significant differences between on and off haloperidol-treatment conditions. When patients returned to haloperidol treatment after relapse, the plasma TAS remained fairly constant and was not significantly different from the same individuals during haloperidol-stabilization or drug-free periods. These findings are indicative of an impaired antioxidant defense system, not attributable to neuroleptic treatment, and lend further support to the notion that oxidative stress may have a pathophysiological role in schizophrenia.

title: Reduced plasma antioxidants in first-episode patients with schizophrenia
abstract: Recent findings suggest that free radical-mediated pathological processes may underlie membrane deficits that have been observed in schizophrenia. We have previously demonstrated that both total antioxidant status (TAS) and individual antioxidants were significantly reduced in plasma of chronic schizophrenic patients, on and off haloperidol. To test whether reductions in plasma antioxidants are independent of treatment or illness progression, levels of plasma antioxidants were evaluated in 43 patients at first-episode of psychosis, 31 patients meeting DSM-IV criteria for schizophrenia or schizoaffective disorder (reconfirmed after 6 months) and 12 patients with primary mood (bipolar or depressive (BP/D)) disorder, at neuroleptic-naive baseline as well as 40 age- and sex-matched normal controls (NC). The major plasma antioxidants albumin, uric acid and bilirubin were all significantly lower in the first-episode schizophrenic, but not in the BP/D patients, than in normal controls. Factors that can affect these antioxidants-age, gender, diet and smoking-were examined. Body mass index (BMI) was significantly lower in patients, but was not correlated with plasma albumin and bilirubin; there was a trend for a correlation with uric acid in schizophrenic patients. Lower antioxidant levels were independent of smoking status of patients. The present data suggest that a defect in the antioxidant defense system (AODS), which may lead to oxidative damage, occurs early in the course of illness and is independent of treatment effects.

title: Oxidative stress, α-tocopherol, ascorbic acid and reduced glutathione status in schizophrenics
abstract: A disturbance in the antioxidant defense system including α-tocopherol, ascorbic acid and reduced glutahtione metabolism due to free radical induced oxidative injury has been implicated in various neuro-psychiatric disorders. The roles of these antioxidants, changes in their blood levels and correlation with oxidative stress were studied in a common psychiatric illness Schizophrenia. Fifty-eight subjects of either sex ranging in age from 18–60 years divided into two age groups (≤40 and >40 years) diagnosed for schizophrenia, and forty age and sex-matched normal subjects as controls were included in the study. Blood samples were analyzed for malondialdehyde (MDA), α-tocopherol, total ascorbic acid (TAA), dehydro ascorbic acid (DHAA), reduced ascorbic acid (RAA), leucocyte ascorbic acid (LAA) and reduced glutathione (GSH). A decrease in the levels of α-tocopherol, total ascorbic acid and reduced glutathione was found in schizophrenics compared to normal controls. Further a significant rise in oxidative stress and decreased antioxidant status was observed in the chronic stage of schizophrenia as compared to those in acute condition. A significant rise in dehydroascorbic acid with concomitant fall in reduced ascorbic acid suggests scavenging action of ascorbic acid and its utilization with increased oxidative stress as indicated by high blood malondialdehyde levels. Leucocyte ascorbic acid, a better index of ascorbic acid status was also found to be reduced in schizophrenics, suggesting depletion of body stores of ascorbic acid and the condition worsened with advancing age.

title: Mitochondrial activity and oxidative stress markers in peripheral blood mononuclear cells of patients with bipolar disorder, schizophrenia, and healthy subjects.
abstract: Evidence suggests that mitochondrial dysfunction is involved in the pathophysiology of psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD). However, the exact mechanisms underlying this dysfunction are not well understood. Impaired activity of electron transport chain (ETC) complexes has been described in these disorders and may reflect changes in mitochondrial metabolism and oxidative stress markers. The objective of this study was to compare ETC complex activity and protein and lipid oxidation markers in 12 euthymic patients with BD type I, in 18 patients with stable chronic SZ, and in 30 matched healthy volunteers. Activity of complexes I, II, and III was determined by enzyme kinetics of mitochondria isolated from peripheral blood mononuclear cells (PBMCs). Protein oxidation was evaluated using the protein carbonyl content (PCC) method, and lipid peroxidation, the thiobarbituric acid reactive substances (TBARS) assay kit. A significant decrease in complex I activity was observed (p = 0.02), as well as an increase in plasma levels of TBARS (p = 0.00617) in patients with SZ when compared to matched controls. Conversely, no significant differences were found in complex I activity (p = 0.17) or in plasma TBARS levels (p = 0.26) in patients with BD vs. matched controls. Our results suggest that mitochondrial complex I dysfunction and oxidative stress play important roles in the pathophysiology of SZ and may be used in potential novel adjunctive therapy for SZ, focusing primarily on cognitive impairment and disorder progression.

title: Altered antioxidant defense system in clinically stable patients with schizophrenia and their unaffected siblings
abstract: To determine Red Blood Cell (RBC) antioxidant enzyme activities and plasma Thiobarbituric Acid Reactive Substances (TBARS) in clinically stable patients with schizophrenia and their unaffected siblings.



A case-control study carried out on three groups: 60 schizophrenic patients treated with neuroleptics, 33 of their unaffected siblings and 30 healthy controls with no family psychiatric history. Biological markers were measured on fasting patients after a period of tobacco abstinence: RBC antioxidant enzyme activities - superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) - by spectrophotometry and plasma levels of TBARS by spectrofluorimetry.



RBC SOD and CAT activities were significantly lower in schizophrenic patients and their unaffected siblings compared to the control group (P<0.001). Schizophrenic patients also had significantly lower RBC GSH-Px activity than controls (P=0.03), whereas their unaffected siblings had significantly higher RBC GSH-Px activity than controls (P=0.04). Plasma TBARS were higher in schizophrenic patients than their unaffected siblings: 2.1+/-0.8 micromol/l vs. 1.7+/-0.6 micromol/l (P=0.06).



Our results showed a decrease in antioxidant enzyme activities and an increase in lipid peroxidation confirming the existence of oxidative stress in schizophrenic patients treated with neuroleptics. Additionally, this suggests that the increase in GSH-Px activity in unaffected siblings would be a protective mechanism against oxidative stress and damage. Other studies are necessary to confirm these findings.

title: Oxidative stress in blood platelets from schizophrenic patients
abstract: Oxidative stress in blood platelets is observed in various diseases, including neuropsychiatric disorders. The aim of our study was to evaluate oxidative stress in blood platelets from patients with schizophrenic disorders by measuring the activity of the platelet antioxidative enzyme, superoxide dismutase (SOD), concomitant with the level of thiobarbituric acid reactive species (TBARS). In blood platelets obtained from schizophrenic patients (with paranoid schizophrenia according to DSM-IV criteria) and from healthy volunteers the level of reactive oxygen species was also measured via chemiluminescence. In resting blood platelets from schizophrenic patients the chemiluminescence was higher than in platelets from control subjects (P < 0.05), but in thrombin-activated platelets an increase (about 53%) of chemiluminescence was observed, however this increase was lower than in thrombin-stimulated platelets from healthy subjects (101.5%). The results indicate that in platelets from schizophrenic patients generation of reactive oxygen species is enhanced. Moreover, we observed that SOD activity in blood platelets from schizophrenic patients was significantly lower than in control platelets and that a correlation exists between increased lipid peroxidation and inhibition of the activity of this antioxidative enzyme in schizophrenic platelets.

title: Elevated serum superoxide dismutase and thiobarbituric acid reactive substances in different phases of bipolar disorder and in schizophrenia
abstract: There is an increasing body of evidence suggesting that oxidative stress may play a role in the pathophysiology of both schizophrenia (SZ) and bipolar disorder (BD).



We compared the antioxidant enzyme, serum superoxide dismutase (SOD) and the lipid peroxidation product, thiobarbituric acid reactive substances (TBARS) as assessed in depressed (N=21), manic (N=32) and euthymic (N=31) bipolar patients, and in chronically medicated patients with schizophrenia (N=97), all fulfilling DSM-IV diagnostic criteria, and a group of healthy controls (N=32).



Serum SOD (U/mg protein) activity was significantly increased (p<0.001) in manic (7.44+/-3.88) and depressed (6.12+/-4.64) BD patients and SZ (9.48+/-4.51) when compared to either controls (1.81+/-0.63) or euthymic (2.75+/-1.09) BD patients. TBARS (mol/L) levels were significantly higher in the SZ group (4.95+/-1.56, p=0.016), bipolar euthymic (6.36+/-1.46, p<0.001), bipolar manic (7.54+/-1.74, p<0.001), and bipolar depressed patients (5.28+/-1.54, p=0.028) compared to controls (3.96+/-1.51).



Our findings show increased SOD activity in SZ, as well as in depressed and manic bipolar patients, but not in euthymic BD subjects. This suggests a dysregulation in oxidative defenses in both disorders. It is likely that such changes reflect state changes in bipolar disorder. It is possible that this is a compensatory response to the oxidative stress that occurs in the acute phase of bipolar episodes. TBARS results show increases in lipid peroxidation in mania. TBARS levels in SZ and in euthymic as well as depressed individuals with BD were higher than in controls. This suggests persistent increases in SZ, which may reflect ongoing symptomatology or treatment, and a state dependent gradient in BD, with greatest oxidative stress in mania. These data support oxidative biology as both a key component of the pathophysiology of both BD and SZ, and the use of agents that modulate oxidative biology as a promising avenue for intervention in both disorders.

title: Towards a classification of biomarkers of neuropsychiatric disease: from encompass to compass
abstract: There is currently considerable imprecision in the nosology of biomarkers used in the study of neuropsychiatric disease. The neuropsychiatric field lags behind others such as oncology, wherein, rather than using 'biomarker' as a blanket term for a diverse range of clinical phenomena, biomarkers have been actively classified into separate categories, including prognostic and predictive tests. A similar taxonomy is proposed for neuropsychiatric diseases in which the core biology remains relatively unknown. This paper divides potential biomarkers into those of (1) risk, (2) diagnosis/trait, (3) state or acuity, (4) stage, (5) treatment response and (6) prognosis, and provides illustrative exemplars. Of course, biomarkers rely on available technology and, as we learn more about the neurobiological correlates of neuropsychiatric disorders, we will realize that the classification of biomarkers across these six categories can change, and some markers may fit into more than one category.

title: Meta-Analysis of Oxidative Stress in Schizophrenia
abstract: Schizophrenia is associated with impaired antioxidant defense, including abnormal serum, plasma, and red blood cell (RBC) oxidative stress parameters. We performed a meta-analysis of these associations, considering the effect of clinical status and antipsychotic treatment after an acute exacerbation of psychosis.



We identified articles by searching PubMed, PsychInfo, and Institute for Scientific Information, and the reference lists of identified studies.



Forty-four studies met the inclusion criteria. Total antioxidant status seemed to be a state marker, because levels were significantly decreased in cross-sectional studies of serum and plasma in first-episode psychosis (FEP) and significantly increased in longitudinal studies of antipsychotic treatment for acute exacerbations of psychosis (p < .01 for each). The RBC catalase and plasma nitrite seemed to be state-related markers, because levels in cross-sectional studies were significantly decreased in FEP (p < .01) and significantly increased in stable outpatients (p = .01). In contrast, RBC superoxide dismutase seemed to be a trait marker for schizophrenia, because levels in cross-sectional studies were significantly decreased in acutely relapsed inpatients, FEP, and stable outpatients (p < .01 for each).



Oxidative stress abnormalities in FEP suggest an effect that might be independent of antipsychotic medications. Although some parameters (total antioxidant status, RBC catalase, and plasma nitrite) might be state markers for acute exacerbations of psychosis, others (RBC superoxide dismutase) might be trait markers; however, more longitudinal studies are needed. Our findings suggest that oxidative stress might serve as a potential biomarker in the etiopathophysiology and clinical course of schizophrenia.

title: Molecular links between mitochondrial dysfunctions and schizophrenia
abstract: Schizophrenia is a complex neuropsychiatric disorder with both neurochemical and neurodevelopmental components in the pathogenesis. Growing pieces of evidence indicate that schizophrenia has pathological components that can be attributable to the abnormalities of mitochondrial function, which is supported by the recent finding suggesting mitochondrial roles for Disrupted-in-Schizophrenia 1 (DISC1). In this minireview, we briefly summarize the current understanding of the molecular links between mitochondrial dysfunctions and the pathogenesis of schizophrenia, covering recent findings from human genetics, functional genomics, proteomics, and molecular and cell biological approaches.

title: Mitochondrial dysfunction in schizophrenia: a possible linkage to dopamine
abstract: Mitochondria are not only the principal source of high energy intermediates, but play an important role in intracellular calcium buffering, are main producers of reactive oxygen species, and are the source of pro‐ and antiapoptotic key factors. Moreover, the mitochondria are of a ubiquitous nature and the respiratory chain has a dual genetic basis, i.e. the mitochondrial and the nuclear DNAs. Thus mitochondrial impairment could provide an explanation for the tremendous heterogeneity of clinical and pathological manifestations in schizophrenia. This article reviews several independent lines of evidence that suggest an involvement of mitochondrial dysfunction in schizophrenia. Among them are altered cerebral energy metabolism, mitochondrial hypoplasia, dysfunction of the oxidative phosphorylation system and altered mitochondrial related gene expression. In addition, the interaction between dopamine, a predominant etiological factor in schizophrenia, and mitochondrial respiration is considered as a possible mechanism underlying the hyper‐ and hypo‐activity cycling in schizophrenia. Understanding the role of mitochondria in schizophrenia may encourage novel treatment approaches, the identification of candidate genes and new insights into the pathophysiology and etiology of the disorder.

title: Electron microscopy of oligodendroglia in severe mental illness
abstract: Qualitative electron microscopy was performed to verify whether brain pathology in schizophrenia and bipolar disorder is associated with alterations of oligodendroglial cells and myelinated fibers. Ultrastructural signs of apoptosis and necrosis of oligodendroglial cells were found in the prefrontal area 10 and the caudate nucleus in both schizophrenia and bipolar disorder. Damage of myelin sheath lamellae, with the formation of concentric lamellar bodies, were detected in both brain structures in schizophrenia. There was also a significant decrease in the area of the nucleus and the volume density of mitochondria in oligodendrogliocytes in the caudate nucleus and in the prefrontal cortex in schizophrenia, as compared to normal controls. Volume density of heterochromatin was significantly increased (+14%) in the caudate nucleus in schizophrenia. The density of concentric lamellar bodies (as an indicator of damage of myelinated fibers) was dramatically increased (4.5-fold) in the caudate nucleus in schizophrenia, as compared to controls, and was positively correlated with volume density of heterochromatin. Multiple regression analysis and analysis of covariance demonstrated that these changes could not be explained by the effects of postmortem delay, age, neuroleptic medication, or gender. Pathology of oligodendroglia might be an essential feature of severe mental disorders.

title: Ultrastructural abnormalities of astrocytes in the hippocampus in schizophrenia and duration of illness: a postortem morphometric study.
abstract: There is a lot of evidence of astrocytic dysfunction in schizophrenia. We performed an electron microscopic morphometric study of astrocytes in the CA3 hippocampal region in 19 cases of schizophrenia and 16 normal controls. No significant group differences were found in cell size, volume fraction (Vv) and area density (Na) of mitochondria and lipofuscin granules. Young control subjects (<50 years old) had significantly lower area of cell, nucleus and cytoplasm and higher Vv and Na of mitochondria than old controls (>50 years old), and young and old schizophrenic cases. No significant differences between young and old schizophrenic subgroups were found. Vv and Na of mitochondria correlated negatively (r=-0.66, r=-0.72, P<0.01) and Vv of lipofuscin granules correlated positively (r=0.72, P=0.001) with duration of illness. These parameters did not correlate with age in controls and in schizophrenic subjects. Both Vv and Na of mitochondria were significantly lower in the subgroup of cases with duration of disease of>21 years. than in the control group and in the subgroup of cases with duration of illness of<21 years (P<0.01). The data suggest progressive disturbances of astrocyte function due to the deficit of mitochondria in schizophrenia.

title: QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.
abstract: Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.

title: Perturbation in Mitochondrial Network Dynamics and in Complex I Dependent Cellular Respiration in Schizophrenia
abstract: Mitochondria have been suggested to be involved in the pathology of bipolar disorder (BD) and schizophrenia. However, the mechanism underlying mitochondrial dysfunction is unclear. Mitochondrial network dynamics, which reflects cellular metabolic state, is important for embryonic development, synapse formation, and neurodegeneration. This study aimed to investigate mitochondrial network dynamics and its plausible association with abnormal cellular oxygen consumption in schizophrenia.



Viable Epstein-Barr virus (EBV)-transformed lymphocytes (lymphoblastoids) from DSM-IV diagnosed patients with schizophrenia (n = 17), BD (n = 15), and healthy control subjects (n = 15) were assessed for mitochondrial respiration, mitochondrial dynamics, and relevant protein levels by oxygraph, confocal microscopy, and immunoblotting, respectively.



Respiration of schizophrenia-derived lymphoblastoids was significantly lower compared with control subjects, and was twice as sensitive to dopamine (DA)-induced inhibition. Unlike DA, haloperidol inhibited complex I-driven respiration to a similar extent in both schizophrenia and the control cells. Both drugs interact with complex I but at different sites. At the site of DA interaction, we found alterations in protein levels of three subunits of complex I in schizophrenia. In addition, we observed structural and connectivity perturbations in the mitochondrial network, associated with alterations in the profusion protein OPA1, which was similarly reduced in schizophrenia prefrontal cortex specimens. None of these alterations were observed in the BD cells, which were similar to control cells.



We show impaired mitochondrial network dynamics associated with reduced cellular respiration and complex I abnormalities in schizophrenia but not in BD. If these findings represent disease-specific alterations, they may become an endophenotype biomarker for schizophrenia.

title: State-dependent alterations in mitochondrial complex I activity in platelets: a potential peripheral marker for schizophrenia
abstract: Schizophrenia, the most severe psychiatric disorder, is characterized by heterogeneity of clinical signs, often categorized into positive and negative symptoms. Among a wide array of competing biological mechanisms, altered cerebral energy metabolism and mitochondrial dysfunction have been suggested to play an important role in the pathophysiology of schizophrenia. In this study we investigated mitochondrial complex I in platelets of 113 schizophrenic patients divided into three groups (acute psychotic episode, chronic active state and residual schizophrenia) and 37 control subjects. Complex I was analysed at the level of enzymatic activity, mRNA and protein levels by enzyme kinetics, RT-PCR and Western blot analyses, respectively. Complex I activity in platelets of schizophrenic patients altered with disease state presenting high specificity and sensitivity. Thus, increased activity was associated with psychotic symptomology, while its decrease was observed in patients with residual schizophrenia. The relationship between the clinical state and complex I activity in schizophrenia was further supported by its positive correlation with the severity of patients’ positive symptoms assessed by clinical ratings. In addition, similar alterations were observed at the levels of mRNA and protein of the 24- and 51-kDa iron-sulfur flavoprotein subunits of the complex. Taken together these results point to the potential of platelet complex I to turn into a reliable novel marker for schizophrenia. At present, definitive diagnosis depends only on descriptive behavioral and symptomatic information, therefore a peripheral measurable specific marker will contribute to diagnosis and monitoring of the disease.

title: Sp1 Expression Is Disrupted in Schizophrenia; A Possible Mechanism for the Abnormal Expression of Mitochondrial Complex I Genes, NDUFV1 and NDUFV2
abstract: The prevailing hypothesis regards schizophrenia as a polygenic disease, in which multiple genes combine with each other and with environmental stimuli to produce the variance of its clinical symptoms. We investigated whether the ubiquitous transcription factor Sp1 is abnormally expressed in schizophrenia, and consequently can affect the expression of genes implicated in this disorder. Principal Findings mRNA of Sp1 and of mitochondrial complex I subunits (NDUFV1, NDUFV2) was analyzed in three postmortem brain regions obtained from the Stanley Foundation Brain Collection, and in lymphocytes of schizophrenic patients and controls. Sp1 role in the transcription of these genes was studied as well. Sp1 was abnormally expressed in schizophrenia in both brain and periphery. Its mRNA alteration pattern paralleled that of NDUFV1 and NDUFV2, decreasing in the prefrontal cortex and the striatum, while increasing in the parieto-occipital cortex and in lymphocytes of schizophrenic patients as compared with controls. Moreover, a high and significant correlation between these genes existed in normal subjects, but was distorted in patients. Sp1 role in the regulation of complex I subunits, was demonstrated by the ability of the Sp1/DNA binding inhibitor, mithramycin, to inhibit the transcription of NDUFV1 and NDUFV2, in neuroblastoma cells. In addition, Sp1 activated NDUFV2 promoter by binding to its three GC-boxes. Both activation and binding were inhibited by mithramycin.  These findings suggest that abnormality in Sp1, which can be the main activator/repressor or act in combination with additional transcription factors and is subjected to environmental stimuli, can contribute to the polygenic and clinically heterogeneous nature of schizophrenia.

title: Increased mitochondrial complex I activity in platelets of schizophrenic patients.
abstract: It is believed that dopamine and alterations of energy metabolism in cortical and subcortical structures are involved in the pathophysiology of schizophrenia. Recently, we and others have shown that dopamine may affect energy metabolism by interacting with mitochondrial complex I activity in rats both in vivo and in vitro. In this study activity of complexes I and IV was assessed in mitochondria isolated from blood platelet of schizophrenic patients and compared to patients with affective disorders and healthy control subjects. Seventy-seven in-patients who met DSM-IV criteria for schizophrenia (in acute exacerbation), bipolar disorder depressed type (BP), or recurrent major depressive disorder (MDD) and 24 control subjects participated in the study. A highly significant increase (240%, p < 0.001) in complex I activity but not in complex IV, was detected in medicated and unmedicated schizophrenic patients compared to controls. No such change was observed in patients with affective disorders. The data demonstrate a specific and selective, alteration in platelet complex I activity in schizophrenic patients, which is not related to medication. If this abnormality in platelet mitochondria reflects brain alterations, it may further support the relevance of alterations in energy metabolism to the pathophysiology of schizophrenia. Finally in the lack of any clinically relevant biological marker for schizophrenia, complex I activity in platelets might become a useful peripheral marker for this disorder.

title: Neuroanatomical Pattern of Mitochondrial Complex I Pathology Varies between Schizophrenia, Bipolar Disorder and Major Depression
abstract: Mitochondrial dysfunction was reported in schizophrenia, bipolar disorderand major depression. The present study investigated whether mitochondrial complex I abnormalities show disease-specific characteristics. Principal Findings mRNA and protein levels of complex I subunits NDUFV1, NDUFV2 and NADUFS1, were assessed in striatal and lateral cerebellar hemisphere postmortem specimens and analyzed together with our previous data from prefrontal and parieto-occipital cortices specimens of patients with schizophrenia, bipolar disorder, major depression and healthy subjects. A disease-specific anatomical pattern in complex I subunits alterations was found. Schizophrenia-specific reductions were observed in the prefrontal cortex and in the striatum. The depressed group showed consistent reductions in all three subunits in the cerebellum. The bipolar group, however, showed increased expression in the parieto-occipital cortex, similar to those observed in schizophrenia, and reductions in the cerebellum, yet less consistent than the depressed group.  These results suggest that the neuroanatomical pattern of complex I pathology parallels the diversity and similarities in clinical symptoms of these mental disorders.

title: Mitochondrial complex i subunits expression is altered in schizophrenia: a postmortem study
abstract: Several independent lines of evidence indicate mitochondrial dysfunction in schizophrenia in the brain and periphery, including mitochondrial hypoplasia, dysfunction of the oxidative phosphorylation system, and altered mitochondrial-related gene expression.



In this study, three subunits of mitochondrial complex I were analyzed at the level of mRNA and protein in postmortem brain specimens from the prefrontal and the ventral parietooccipital cortex of patients with schizophrenia, major depression, bipolar disorder, and normal control subjects.



Both mRNA and protein levels of the 24-kDa and 51-kDa subunits of complex I were significantly decreased in the prefrontal cortex, but increased in the ventral parietooccipital cortices of schizophrenia patients compared with normal control subjects. In the latter region, protein levels of both subunits were increased in bipolar patients as well, being in line with the significant overlap in clinical symptoms between schizophrenia and bipolar patients. No change was observed in the 75-kDa subunit expression in the prefrontal cortex.



The schizophrenia-specific reduction in complex I subunits in the prefrontal cortex is consistent with one of schizophrenia's most prominent deficits, namely, hypofrontality, thus further supporting the hypothesis of mitochondrial dysfunction in this disorder. The abnormal, bidirectional expression of complex I in various brain regions, rather than in a circumscribed area, supports the idea of impaired cerebral circuitry in schizophrenia.

title: Mitochondrial dysfunction in schizophrenia: Pathways, mechanisms and implications
abstract: Mitochondria play a critical role in regulating cellular functions including bioenergetics, calcium homeostasis, redox signalling, and apoptotic cell death. Mitochondria are also essential to many aspects of neurodevelopment and neuronal functions. However, mitochondrial impairment may affect bioenergetics in the developing brain and alter critical neuronal processes leading to neurodevelopmental abnormalities. Schizophrenia is a chronic and severe neuropsychiatric disorder of neurodevelopmental origin. Immuno-inflammatory pathway is one of the widely appreciated mechanisms that has consistently been implicated in the neurodevelopmental origin of schizophrenia. However, the source of inflammation and the underlying neurobiological mechanisms leading to schizophrenia are yet to be fully ascertained. Recent understanding reveals that perturbation of mitochondrial network dynamics might lead to various nervous system disorders with inflammatory pathologies. Mitochondrial deficit, altered redox balance and chronic low-grade inflammation are evident in schizophrenia. It is hypothesized that oxidative/nitrosative stress responses due to mitochondrial dysfunctions might activate immuno-inflammatory pathways and subsequently lead to neuroprogressive changes in schizophrenia. Herein, we summarise the current understanding of molecular links between mitochondrial dysfunctions and pathogenesis of schizophrenia based on evidence from genomics, proteomics and imaging studies, which together support a role for mitochondrial impairment in the pathogenetic pathways of schizophrenia.

title: Evidence for a mitochondrial oxidative phosphorylation defect in brains from patients with schizophrenia
abstract: In-vivo imaging studies and post-mortem studies have demonstrated an impairment of energy metabolism in brains of patients with schizophrenia. Decreased oxidative metabolism has been consistently documented in the frontal lobes. However, the biochemical basis of these changes is unclear. The changes could be caused by reduced requirement of the cells for metabolic energy or an abnormality in energy generation. Neurons generate energy through the respiratory chain in the mitochondria. The respiratory chain consists of five enzyme complexes (I-V). The purpose of the present study was to assess mitochondrial function and test the hypothesis of an underlying oxidative phosphorylation defect in schizophrenia. We analysed spectrophotometrically post-mortem brain specimens of frontal cortex, temporal cortex, basal ganglia, and cerebellum of 12 patients who met the DSM-IV criteria for schizophrenia and of 13 healthy controls for the specific activities of respiratory chain enzymes in the mitochondria. The major finding was that the activity of complex IV was significantly reduced in the frontal cortex (40.9+/-6.7 vs. 87.3+/-12, P=0.003) and in the temporal cortex (39.5+/-6.8 vs. 78+/-10.8, P=0.006) of schizophrenics. In addition, the activity of complexes I+III was significantly reduced in the temporal cortex (2.2+/-0.6 vs. 4.4+/-0.5, P=0.01) and basal ganglia (1.6+/-0.5 vs. 3.4+/-0.3, P=0.015) in schizophrenia. All other enzyme activities showed no differences to healthy controls. The results confirm a defect of oxidative phosphorylation in brains from patients with schizophrenia, which may contribute to impaired energy generation.

title: Glucose/mitochondria in neurological conditions.
abstract: Impairments of glucose and mitochondrial function are important causes of brain dysfunction and therefore of brain disease. Abnormalities have been found in association with disease of the nervous system in most of the components of glucose/mitochondrial metabolism. In many, molecular genetic abnormalities have been defined. Brain glucose oxidation is abnormal in common diseases of the nervous system, including Alzheimer disease and other dementias, Parkinson disease, delirium, probably schizophrenia and other psychoses, and of course cerebrovascular disease. Defects in a single component and even a single mutation can be associated with different clinical phenotypes. The same clinical phenotype can result from different genotypes. The complex relationship between biological abnormality in brain glucose utilization and clinical disorder is similar to that in other disorders that have been intensively studied at the genetic level. Genes for components of the pathways of brain glucose oxidation are good candidate genes for disease of the brain. Preliminary data support the proposal that treatments to normalize abnormalities in brain glucose oxidation may benefit many patients with common brain diseases.

title: Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation
abstract: BackgroundSchizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing.MethodsWe performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area – BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease.ResultsOur analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex.ConclusionOur data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

title: Proteome analysis of the thalamus and cerebrospinal fluid reveals glycolysis dysfunction and potential biomarkers candidates for schizophrenia.
abstract: Schizophrenia (SCZ) is the result of DNA alterations and environmental factors, which together lead to differential protein expression and ultimately to the development of the illness. The diagnosis is based on clinical symptoms, and the molecular background of SCZ is not completely understood. The thalamus, whose dysfunction has been associated with SCZ based in diverse lines of evidences, plays for instance a pivotal role in the central nervous system as a relay center by re-distributing auditory and visual stimuli from diverse brain regions to the cerebral cortex. We analyzed the proteome of postmortem mediodorsal thalamus (MDT) samples from 11 SCZ patients and 8 non-SCZ individuals by using quantitative shotgun-mass spectrometry and two-dimensional gel electrophoresis. Our analyses identified 551 proteins, 50 of which showed significant differential expression. The main pathways affected by the differentially expressed proteins include energy metabolism, oligodendrocyte metabolism, and cytoskeleton assembly. The potential protein biomarkers candidates myelin basic protein and myelin oligodendrocyte protein were validated by Western blot in the MDT samples and also in cerebrospinal fluid from a separate set of samples of 17 first-episode SCZ patients and 10 healthy controls. The differential expression of μ-crystallin, protein kinase C-gamma, and glial fibrillary acidic protein were confirmed in MDT. Because we found several glycolysis enzymes to be differentially expressed, we measured the levels of pyruvate and NADPH and found them to be altered in MDT. The protein changes described here corroborate the importance of myelin/oligodendrocyte and energy metabolism in SCZ and highlight new potential biomarkers candidates that may contribute to the understanding of the pathogenesis of this complex disease.

title: Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress
abstract: The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcriptomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcriptional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.

title: Reduced phosphodiesters and high-energy phosphates in the frontal lobe of schizophrenic patients: a 31P chemical shift spectroscopic-imaging study
abstract: (31)Phosphorous magnetic resonance spectroscopy has been widely used to evaluate schizophrenic patients in comparison to control subjects, because it allows the investigation of both phospholipid and energy metabolism in vivo; however, the results achieved so far are inconsistent. Chemical shift imaging (CSI) has the advantage that instead of only one or a few preselected voxels the tissue of a whole brain slice can be examined. The aim of the present investigation was to determine whether the results of previous studies of our group, showing that phosphodiesters (PDE) are decreased in the frontal lobe of schizophrenic patients as compared to control subjects, might be confirmed in an independent unmedicated patient sample using the CSI technique.



A carefully selected new cohort including 11 neuroleptic-free schizophrenic patients and 11 age- and gender-matched healthy control subjects was recruited. CSI was applied and an innovative analysis method for CSI data based on a general linear model was used.



PDE, phosphocreatine, and adenosine triphosphate (ATP) were found to be significantly decreased in the frontal lobe of patients with schizophrenia.



Because PDE was decreased in schizophrenic patients, the membrane phospholipid hypothesis of schizophrenia could not be corroborated. Further results indicate decreased ATP production in the frontal lobe of patients with schizophrenia.

title: New evidence for the involvement of mitochondrial inheritance in schizophrenia: results from a cross-sectional study evaluating the risk of illness in relatives of schizophrenia patients.
abstract: One of the hypotheses about the genetic factors that contribute to schizophrenia involves mitochondrial DNA (mtDNA), an approximately 16,569-base pair molecule inherited only from the mother. If this hypothesis were true, one would expect a higher frequency of schizophrenia among matrilineal relatives who share mtDNA with a schizophrenia patient than among relatives who do not. This article reports the risk of presenting with schizophrenia, other psychiatric disorders, and conditions related to mitochondrial disorders in relatives who share mtDNA with a schizophrenia patient versus those who do not.



We interviewed 100 schizophrenia patients (DSM-IV criteria) and 147 of their first-degree relatives from November 2007 to November 2009 to collect clinical data from patients and from both sides of each patient's pedigree. The study was conducted at of a psychiatric teaching hospital in Reus, Spain. Contingency tables were established, and odds ratios were calculated to estimate relative risk.



Relatives who shared mtDNA with a schizophrenia patient had a higher risk of presenting with schizophrenia than those who did not share mtDNA (odds ratio [OR] = 3.05; 95% CI, 1.65-5.72; P < .001). Female but not male relatives who shared mtDNA with a schizophrenia patient also had a higher risk of unipolar depression (OR =10.19; 95% CI, 4.07-32.80; P < .001), panic attack (OR = 15.52; 95% CI, 2.41-643.6; P < .001), and other anxiety disorders (OR = 4.14; 95% CI, 1.84-9.71; P < .001). Some conditions frequently associated with mitochondrial disorders were also more frequent among female relatives who shared mtDNA with a schizophrenia patient than among those who did not.



The results of this study support the hypothesis that mtDNA may be involved in schizophrenia. In females, mtDNA could also be involved in the development of other psychiatric and nonpsychiatric conditions. Further studies are needed to confirm the role of mtDNA in psychiatric disorders.

title: Mechanistic explanations how cell-mediated immune activation, inflammation and oxidative and nitrosative stress pathways and their sequels and concomitants play a role in the pathophysiology of unipolar depression
abstract: This paper reviews that cell-mediated-immune (CMI) activation and inflammation contribute to depressive symptoms, including anhedonia; anxiety-like behaviors; fatigue and somatic symptoms, e.g. illness behavior or malaise; and mild cognitive impairment (MCI). These effects are in part mediated by increased levels of pro-inflammatory cytokines (PICs), e.g. interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF)α, and Th-1-derived cytokines, such as IL-2 and interferon (IFN)γ. Moreover, new pathways, i.e. concomitants and sequels of CMI activation and inflammation, were detected in depression: (1) Induction of indoleamine 2,3-dioxygenase (IDO) by IFNγ and some PICs is associated with depleted plasma tryptophan, which may interfere with brain 5-HT synthesis, and increased production of anxiogenic and depressogenic tryptophan catabolites. (2) Increased bacterial translocation may cause depression-like behaviors by activating the cytokine network, oxidative and nitrosative stress (O&NS) pathways and IDO. (3) Induction of O&NS causes damage to membrane ω3 PUFAs, functional proteins, DNA and mitochondria, and autoimmune responses directed against intracellular molecules that may cause dysfunctions in intracellular signaling. (4) Decreased levels of ω3 PUFAs and antioxidants, such as coenzyme Q10, glutathione peroxidase or zinc, are associated with an increased inflammatory potential; more oxidative damage; the onset of specific symptoms; and changes in the expression or functions of brain 5-HT and N-methyl-d-aspartate receptors. (5) All abovementioned factors cause neuroprogression, that is a combination of neurodegeneration, neuronal apoptosis, and lowered neurogenesis and neuroplasticity. It is concluded that depression may be the consequence of a complex interplay between CMI activation and inflammation and their sequels/concomitants which all together cause neuroprogression that further shapes the depression phenotype. Future research should employ high throughput technologies to collect genetic and gene expression and protein data from patients with depression and analyze these data by means of systems biology methods to define the dynamic interactions between the different cell signaling networks and O&NS pathways that cause depression.

title: Age-specific familial risks of psychotic disorders and schizophrenia: A nation-wide epidemiological study from Sweden
abstract: This study analyzed men and women separately by age at hospital diagnosis of psychotic disorder or schizophrenia and by maternal or paternal disease after taking several possible confounders into account.



The Multigeneration Register, in which all men and women born in Sweden from 1932 onwards are registered together with their parents, was linked to hospital data. This yielded 21,199 male and 19,029 female cases of psychotic disorders in addition to 12,799 paternal and 23,021 maternal cases of psychotic disorders (including schizophrenia). Standardized incidence ratios (SIRs) were calculated as the ratio of observed and expected number of cases among men and women with mothers and/or fathers affected by psychotic disorders or schizophrenia, compared with men and women whose mothers and/or fathers were not affected by psychotic disorders or schizophrenia.



The overall significant SIRs among men and women with a mother, father or both parents hospitalized for psychotic disorder varied between 2.86 and 20.30. Maternal transmission of psychotic disorder was stronger than paternal, and the highest SIRs were found in the youngest age groups. Similar results were found when the subgroup schizophrenia was analyzed separately. Maternal or paternal schizophrenia implied higher risks for the offspring than maternal or paternal psychotic disorders.



Hereditary factors have a strong influence on the onset of psychotic disorders and schizophrenia. Young people and individuals with both parents affected by these diseases need special attention as their SIRs were particularly increased.

title: Aberrant tyrosine transport across the fibroblast membrane in patients with schizophrenia--indications of maternal inheritance.
abstract: In previous studies of the present patients with schizophrenia, aberrant tyrosine transport across the fibroblast membrane was found. A low K(m), a kinetic factor indicating high affinity between tyrosine and the binding site at the cell membrane, was found to be associated with poor cognitive functions in patients. The present study aimed at investigating possible relationships between patients with schizophrenia and their first-degree relatives in aberrant tyrosine transport indicating that it may be a biological marker for the genetic susceptibility.



Thirty-three parents, 13 fathers and 20 mothers, from 23 families with a schizophrenic patient agreed to enter the study. They underwent skin biopsies for fibroblast cultivation, neuropsychological and psychiatric investigations and were classified as family history positive or negative. Tyrosine transport kinetics (K(m) and V(max)) were calculated from in vitro trials of gradients of extracellular tyrosine concentrations in fibroblast cultures.



An association between patients with schizophrenia and their mothers were found for a low K(m) indicating maternal inheritance. Mothers displaying a low K(m) performed worse on the neuropsychological tests compared to mothers with normal K(m). Corresponding relationships between a low K(m) and neurocognitive dysfunction had previously been found for the patients.



An aberrant tyrosine transport across plasma membrane may constitute a biological marker for an endophenotype within the schizophrenia spectrum with low cognitive functioning. A plausible mode for genetic transmission is maternal inheritance.

title: A mitochondrial DNA sequence variant associated with schizophrenia and oxidative stress
abstract: We have previously reported a changed mitochondrial (mt) gene expression in brain from patients with schizophrenia [ (1995) 203]; now, we describe the distribution in the mtDNA from lymphocytes of a heteroplasmic sequence variation that was originally found in the mtDNA from the postmortem brain of a patient with schizophrenia. The variant is m.12027T>C and results in the change from isoleucine to threonine at position 423 of the ND4 subunit of NADH-ubiquinone reductase. Using a PCR-RFLP method, we have determined the heteroplasmy as the ratio of variant to total (variant ratio) at m.12027 in 184 controls and 181 patients with schizophrenia as well as 24 postmortem brain samples. The distribution of variants is bimodal having peaks at variant ratios of 0.262 and 0.732. The variant-rich fraction is very significantly associated with schizophrenia in males (47%), while there is only 18% in control males. There are significantly more variant-rich control females (36%) than control males (18%), suggesting that the female population is less sensitive to the presence of a variant in terms of liability to schizophrenia. In variant-rich samples from postmortem brain originating from both sexes, there is an increased superoxide production, suggesting that the variation contributes to oxidative stress. Antioxidant glycosides, such as quercetin rutoside, quench the superoxide production without (in contrast to neuroleptic drugs) interfering with the electron transfer activity of the reductase.

title: Analysis of mitochondrial DNA variants in Japanese patients with schizophrenia.
abstract: To test the hypothesis that mitochondrial DNA (mtDNA) variants contribute to the susceptibility to schizophrenia, we sequenced the entire mtDNAs from 93 Japanese schizophrenic patients. Three non-synonymous homoplasmic variants in subunit six of the ATP synthase (MT-ATP6) gene that were detected only in patients but not in controls were suggested to be slightly deleterious, because (1) their original amino acid residues (AA) were highly conserved and (2) the physicochemical differences between the original and altered AA were relatively high. In addition, we detected three novel heteroplasmic variants that were potentially pathogenic. Although functional analysis is needed, rare variants in the mtDNA may convey susceptibility to schizophrenia.

title: Schizophrenia: maternal inheritance and heteroplasmy of mtDNA mutations.
abstract: Role of mitochondrial pathology in schizophrenia has not been fully clarified. We searched for distinctive variants in mtDNA extracted from the gray matter of postmortem brains and from peripheral blood samples. We screened mtDNA region containing 5 genes encoding subunits of cytochrome c oxidase and ATPases. Polymorphisms not already reported in databases are recorded as unregistered rare variants. Four unregistered, non-synonymous rare variants were detected in 4 schizophrenic samples.  non-synonymous variants were not previously detected in non-psychotic Japanese samples registered in the mtSNP database. These variants may contribute to disease pathophysiology. In one family, compound mutations showed co-segregation with schizophrenia. MtDNA mutations could confer a risk for schizophrenia in the Japanese population, although further analyses are needed.

title: Nitric oxide and mitochondrial biogenesis
abstract: The characteristic structural organization of mitochondria is the product of synthesis of macromolecules within the mitochondria together with the import of proteins and lipids synthesized outside the organelle. Synthetic and import processes are required for mitochondrial proliferation and might also facilitate the growth of pre-existing mitochondria. Recent evidence indicates that these events are regulated in a complex way by several agonists and environmental conditions, through activation of specific signaling pathways and transcription factors. A newly discovered role of this organelle in retrograde intracellular signaling back to the nucleus has also emerged. This is likely to have far-reaching implications in development, aging, disease and environmental adaptation. Generation of nitric oxide (NO) appears to be an important player in these processes, possibly acting as a unifying molecular switch to trigger the whole mitochondrial biogenesis process. High levels of NO acutely inhibit cell respiration by binding to cytochrome c oxidase. Conversely, chronic, smaller increases in NO levels stimulate mitochondrial biogenesis in diverse cell types. NO-induced mitochondrial biogenesis seems to be linked to proliferation and differentiation of normal and tumor cells, as well as in aging.

title: Physiology of nitric oxide in skeletal muscle.
abstract: In the past five years, skeletal muscle has emerged as a paradigm of "nitric oxide" (NO) function and redox-related signaling in biology. All major nitric oxide synthase (NOS) isoforms, including a muscle-specific splice variant of neuronal-type (n) NOS, are expressed in skeletal muscles of all mammals. Expression and localization of NOS isoforms are dependent on age and developmental stage, innervation and activity, history of exposure to cytokines and growth factors, and muscle fiber type and species. nNOS in particular may show a fast-twitch muscle predominance. Muscle NOS localization and activity are regulated by a number of protein-protein interactions and co- and/or posttranslational modifications. Subcellular compartmentalization of the NOSs enables distinct functions that are mediated by increases in cGMP and by S-nitrosylation of proteins such as the ryanodine receptor-calcium release channel. Skeletal muscle functions regulated by NO or related molecules include force production (excitation-contraction coupling), autoregulation of blood flow, myocyte differentiation, respiration, and glucose homeostasis. These studies provide new insights into fundamental aspects of muscle physiology, cell biology, ion channel physiology, calcium homeostasis, signal transduction, and the biochemistry of redox-related systems.

title: The Chemical Interplay between Nitric Oxide and Mitochondrial Cytochrome c Oxidase: Reactions, Effectors and Pathophysiology
abstract: Nitric oxide (NO) reacts with Complex I and cytochrome c oxidase (CcOX, Complex IV), inducing detrimental or cytoprotective effects. Two alternative reaction pathways (PWs) have been described whereby NO reacts with CcOX, producing either a relatively labile nitrite-bound derivative (CcOX-NO2  −, PW1) or a more stable nitrosyl-derivative (CcOX-NO, PW2). The two derivatives are both inhibited, displaying different persistency and O2 competitiveness. In the mitochondrion, during turnover with O2, one pathway prevails over the other one depending on NO, cytochrome c 2+ and O2 concentration. High cytochrome c 2+, and low O2 proved to be crucial in favoring CcOX nitrosylation, whereas under-standard cell-culture conditions formation of the nitrite derivative prevails. All together, these findings suggest that NO can modulate physiologically the mitochondrial respiratory/OXPHOS efficiency, eventually being converted to nitrite by CcOX, without cell detrimental effects. It is worthy to point out that nitrite, far from being a simple oxidation byproduct, represents a source of NO particularly important in view of the NO cell homeostasis, the NO production depends on the NO synthases whose activity is controlled by different stimuli/effectors; relevant to its bioavailability, NO is also produced by recycling cell/body nitrite. Bioenergetic parameters, such as mitochondrial ΔΨ, lactate, and ATP production, have been assayed in several cell lines, in the presence of endogenous or exogenous NO and the evidence collected suggests a crucial interplay between CcOX and NO with important energetic implications.

title: Nitric oxide and mitochondrial signaling: from physiology to pathophysiology.
abstract: Nitric oxide (NO) has been known for many years to bind to cytochrome C oxidase, the terminal acceptor in the mitochondrial electron transport chain, in competition with oxygen. This interaction may be significant in vivo and explain some of the biological actions of NO. In this article we review the evidence showing that binding of NO to cytochrome C oxidase elicits intracellular signaling events, including the diversion of oxygen to nonrespiratory substrates and the generation of reactive oxygen species. We discuss findings indicating that these NO-elicited events act as triggers by which mitochondria modulate signal transduction cascades involved in the induction of cellular defense mechanisms and adaptive responses. We also discuss instances in which the effects of NO on the electron transport chain might lead to mitochondrial dysfunction and pathology.

title: Nitric oxide： orchestrating hypoxia regulation through mitochondrial respiration and the endoplasmic reticulum stress response
abstract: Mitochondria have long been considered to be the powerhouse of the living cell, generating energy in the form of the molecule ATP via the process of oxidative phosphorylation. In the past 20 years, it has been recognised that they also play an important role in the implementation of apoptosis, or programmed cell death. More recently it has become evident that mitochondria also participate in the orchestration of cellular defence responses. At physiological concentrations,the gaseous molecule nitric oxide (NO) inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen. This interaction underlies the mitochondrial actions of NO, which range from the physiological regulation of cell respiration, through mitochondrial signalling, to the development of "metabolic hypoxia" - a situation in which, although oxygen is available, the cell is unable to utilise it.

title: Mitochondrial free radical production and cell signaling.
abstract: Mitochondria were classically recognized as the organelles that produce the energy required to drive the endergonic processes of cell life, but now they are considered as the most important cellular source of free radicals, as the main target for free radical regulatory and toxic actions, and as the source of signaling molecules that command cell cycle, proliferation and apoptosis. The progress in the knowledge of mitochondrial functions in the last decades is reviewed. The mitochondrial production of the primary free radicals superoxide anion (O(2)(-)) and nitric oxide (NO), as well as of the termination products H(2)O(2) (hydrogen peroxide) and peroxynitrite (ONOO(-)), is described. A network of intramitochondrial antioxidants consisting of the enzymes Mn-superoxide dismutase and glutathione peroxidase and of the reductants NADH(2), ubiquinol and reduced glutathione, is operative in minimizing the potentially harmful effects of O(2)(-), NO, H(2)O(2) and ONOO(-). Nitric oxide and H(2)O(2) participate in cell signaling, through narrow concentration ranges that signal for opposite cellular situations, i.e., proliferation or apoptosis. A mechanism involving mitogen-activated protein kinases is described. The role of mitochondria in apoptosis is well established through the mitochondrion-dependent pathways of cell death, that includes increased NO production, loss of membrane potential, appearance of dysfunctional mitochondria, cytochrome c release and opening of the voltage-dependent anion channel of the outer membrane.

title: Regulation of Mitochondrial Respiration by Oxygen and Nitric Oxide
abstract: Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1‐ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2−) and peroxynitrite (ONOO−). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. NO exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

title: Does nitric oxide modulate mitochondrial energy generation and apoptosis?
abstract: The physiological role of nitric oxide (NO) in the maintenance of vascular tone, in synaptic transmission and in cellular defence is now firmly established. Recent evidence indicates that NO can also affect mitochondrial function. Here, we review findings indicating that NO — through its interaction with components of the electron-transport chain — might function not only as a physiological regulator of cell respiration, but also to augment the generation of reactive oxygen species by mitochondria, and thereby trigger mechanisms of cell survival or death.

title: Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols.
abstract: NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.

title: Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles.
abstract: Nitric oxide (.NO) released by S-nitrosoglutathione (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective .NO concentration of 0.1 microM, while succinate- and NADH-cytochrome-c reductase activities were half-maximally inhibited at 0.3 microM .NO. Submitochondrial particles treated with .NO (either from GSNO or from a pure solution) showed increased O(-)(2) and H202 production when supplemented with succinate alone, at rates that were comparable to those of control particles with added succinate and antimycin. Rat heart mitochondria treated with .NO also showed increased H2O2 production. Cytochrome spectra and decreased enzymatic activities in the presence of .NO are consistent with a multiple inhibition of mitochondrial electron transfer at cytochrome oxidase and at the ubiquinone-cytochrome b region of the respiratory chain, the latter leading to the increased O2- production. Electrochemical detection showed that the buildup of a .NO concentration from GSNO was interrupted by submitochondrial particles supplemented with succinate and antimycin and was restored by addition of superoxide dismutase. The inhibitory effect of .NO on cytochrome oxidase was also prevented under the same conditions. Apparently, mitochondrial O2- reacts with .NO to form peroxynitrate and, by removing .NO, reactivates the previously inhibited cytochrome oxidase. It is suggested that, at physiological concentrations of .NO, inhibition of electron transfer, .NO-induced O2- production, and ONOO- formation participate in the regulatory control of mitochondrial oxygen uptake.

title: Neurodegeneration and energy metabolism: from chemistry to clinics
abstract: 1 Laboratory of Functional Neurochemistry, Neurological Institute ‘C. Mondino’, Pavia, Italy 2 Signal Transduction Research Group, Neuroscience Research Center of the Charite/Institute for Physiology Humboldt University Berlin,  D-10117, Germany 3 Learning and Memory Research,  1/116, Universitaetsstr 150, Bochum 44780, Germany 4 Department of Neurological Sciences, University of Rome ‘La Sapienza’, Rome I-00185, Italy 5 Department of Biochemical Sciences, University of Rome ‘La Sapienza’, Rome I-00185, Italy All authors have contributed to this work equally *  P Sarti, Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Università di Roma ‘La Sapienza’, Piazzale Aldo Moro 5, Roma I-00185,  þ 39-06-4450291; Fax: þ 39-06-4440062; E-mail: paolo.sarti@uniroma1.it

title: Nitroxia: the pathological consequence of dysfunction in the nitric oxide-cytochrome c oxidase signaling pathway.
abstract: It is now recognized that mitochondria play an integral role in orchestrating the response of the cell to a wide variety of metabolic and environmental stressors. Of particular interest are the interactions of reactive oxygen and nitrogen species with the organelle and their potential regulatory function. The best understood example is the O(2) sensitive binding of NO (nitric oxide) to the heme group in cytochrome c oxidase. We have proposed that this reversible process serves the function of both regulating the formation of hydrogen peroxide from the respiratory chain for the purposes of signal transduction and controlling O(2) gradients in complex organs such as the liver or heart. It now appears that maladaptation in this pathway leads to a mitochondrial dysfunction which has some of the characteristics of hypoxia, such as a deficit in ATP, but occurs in the presence of normal or enhanced levels of O(2). These are the optimal conditions for the formation of reactive nitrogen species (RNS), such as peroxynitrite which lead to the irreversible modification of proteins. We term this unique pathological condition Nitroxia and describe how it may contribute to the pathology of chronic inflammatory diseases using ethanol-dependent hepatotoxicity as an example.

title: Nitric oxide regulation of mitochondrial oxygen consumption II: Molecular mechanism and tissue physiology.
abstract: Nitric oxide (NO) is an intercellular signaling molecule; among its many and varied roles are the control of blood flow and blood pressure via activation of the heme enzyme, soluble guanylate cyclase. A growing body of evidence suggests that an additional target for NO is the mitochondrial oxygen-consuming heme/copper enzyme, cytochrome c oxidase. This review describes the molecular mechanism of this interaction and the consequences for its likely physiological role. The oxygen reactive site in cytochrome oxidase contains both heme iron (a(3)) and copper (Cu(B)) centers. NO inhibits cytochrome oxidase in both an oxygen-competitive (at heme a(3)) and oxygen-independent (at Cu(B)) manner. Before inhibition of oxygen consumption, changes can be observed in enzyme and substrate (cytochrome c) redox state. Physiological consequences can be mediated either by direct "metabolic" effects on oxygen consumption or via indirect "signaling" effects via mitochondrial redox state changes and free radical production. The detailed kinetics suggest, but do not prove, that cytochrome oxidase can be a target for NO even under circumstances when guanylate cyclase, its primary high affinity target, is not fully activated. In vivo organ and whole body measures of NO synthase inhibition suggest a possible role for NO inhibition of cytochrome oxidase. However, a detailed mapping of NO and oxygen levels, combined with direct measures of cytochrome oxidase/NO binding, in physiology is still awaited.

title: Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell.
abstract: The aim of this work is to review the information available on the molecular mechanisms by which the NO radical reversibly downregulates the function of cytochrome c oxidase (CcOX). The mechanisms of the reactions with NO elucidated over the past few years are described and discussed in the context of the inhibitory effects on the enzyme activity. Two alternative reaction pathways are presented whereby NO reacts with the catalytic intermediates of CcOX populated during turnover. The central idea is that at "cellular" concentrations of NO (</= microM), the redox state of the respiratory chain results in the formation of either the nitrosyl- or the nitrite-derivative of CcOX, with potentially different metabolic implications for the cell. In particular, the role played by CcOX in protecting the cell from excess NO, potentially toxic for mitochondria, is also reviewed highlighting the mechanistic differences between eukaryotes and some prokaryotes.

title: The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance
abstract: The four gases, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H2S) and hydrogen cyanide (HCN) all readily inhibit oxygen consumption by mitochondrial cytochrome oxidase. This inhibition is responsible for much of their toxicity when they are applied externally to the body. However, recently these gases have all been implicated, to greater or lesser extents, in normal cellular signalling events. In this review we analyse the chemistry of this inhibition, comparing and contrasting mechanism and discussing physiological consequences. The inhibition by NO and CO is dependent on oxygen concentration, but that of HCN and H2S is not. NO and H2S are readily metabolised by oxidative processes within cytochrome oxidase. In these cases the enzyme may act as a physiological detoxifier of these gases. CO oxidation is much slower and unlikely to be as physiologically important. The evidence for normal physiological levels of these gases interacting with cytochrome oxidase is equivocal, in part because there is little robust data about their steady state concentrations. A reasonable case can be made for NO, and perhaps CO and H2S, inhibiting cytochrome oxidase in vivo, but endogenous levels of HCN seem unlikely to be high enough.

title: Nitric Oxide in Skeletal Muscle: Role on Mitochondrial Biogenesis and Function
abstract: Nitric oxide (NO) has been implicated in several cellular processes as a signaling molecule and also as a source of reactive nitrogen species (RNS). NO is produced by three isoenzymes called nitric oxide synthases (NOS), all present in skeletal muscle. While neuronal NOS (nNOS) and endothelial NOS (eNOS) are isoforms constitutively expressed, inducible NOS (iNOS) is mainly expressed during inflammatory responses. Recent studies have demonstrated that NO is also involved in the mitochondrial biogenesis pathway, having PGC-1α as the main signaling molecule. Increased NO synthesis has been demonstrated in the sarcolemma of skeletal muscle fiber and NO can also reversibly inhibit cytochrome c oxidase (Complex IV of the respiratory chain). Investigation on cultured skeletal myotubes treated with NO donors, NO precursors or NOS inhibitors have also showed a bimodal effect of NO that depends on the concentration used. The present review will discuss the new insights on NO roles on mitochondrial biogenesis and function in skeletal muscle. We will also focus on potential therapeutic strategies based on NO precursors or analogs to treat patients with myopathies and mitochondrial deficiency.

title: Nitric oxide-induced persistent inhibition and nitrosylation of active site cysteine residues of mitochondrial cytochrome-c oxidase in lung endothelial cells.
abstract: Persistent inhibition of cytochrome-c oxidase, a terminal enzyme of the mitochondrial electron transport chain, by excessive nitric oxide (NO) derived from inflammation, polluted air, and tobacco smoke contributes to enhanced oxidant production and programmed cell death or apoptosis of lung cells. We sought to determine whether the long-term exposure of pulmonary artery endothelial cells (PAEC) to pathophysiological concentrations of NO causes persistent inhibition of complex IV through redox modification of its key cysteine residues located in a putative NO-sensitive motif. Prolonged exposure of porcine PAEC to 1 mM 2,2'-(hydroxynitrosohydrazino)-bis-ethanamine (NOC-18; slow-releasing NO donor, equivalent to 1-5 microM NO) resulted in a gradual, persistent inhibition of complex IV concomitant with a reduction in ratios of mitochondrial GSH and GSSG. Overexpression of thioredoxin in mitochondria of PAEC attenuated NO-induced loss of complex IV activities, suggesting redox regulation of complex IV activity. Sequence analysis of complex IV subunits revealed a novel putative NO-sensitive motif in subunit II (S2). There are only two cysteine residues in porcine complex IV S2, located in the putative motif. Immunoprecipitation and Western blot analysis and "biotin switch" assay demonstrated that exposure of PAEC to 1 mM NOC-18 increased S-nitrosylation of complex IV S2 by 200%. Site-directed mutagenesis of these two cysteines of complex IV S2 attenuated NO-increased nitrosylation of complex IV S2. These results demonstrate for the first time that NO nitrosylates active site cysteines of complex IV, which is associated with persistent inhibition of complex IV. NO inhibition of complex IV via nitrosylation of NO-sensitive cysteine residues can be a novel upstream event in NO-complex IV signaling for NO toxicity in lung endothelial cells.

title: S-Nitrosation of Mitochondrial Complex I Depends on Its Structural Conformation*
abstract: Nitric oxide is known to cause persistent inhibition of mitochondrial respiration as a result of S-nitrosation of NADH: ubiquinone oxidoreductase (complex I) (Clementi, E., Brown, G. C., Feelisch, M., and Moncada, S. (1998) ). Little is known about whether such nitrosation occurs in physiological conditions and, if so, what are the possible cellular mechanisms. We have now found that the conformational state (active/deactive transition (Vinogradov, A. D. (1998)  Acta 1364, 169–185)) of mitochondrial complex I is an important factor for the interaction of the enzyme with nitrosothiols and peroxynitrite. Only the deactivated, idle form of complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. In contrast, the active form of the enzyme was insensitive to such treatment. Neither form of complex I was inhibited by nitric oxide itself. Our data suggest that the process of active/deactive transition plays an important role in the regulation of complex I activity and cellular respiration by nitric oxide. The implications of this finding for hypoxic or pathophysiological conditions in vivo are discussed.

title: Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione.
abstract: Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

title: Nitric oxide and peroxynitrite cause irreversible increases in the K(m) for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies.
abstract: Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K(m) for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K(m). These include: treatment of the purified enzyme with peroxynitrite or high (microM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.

title: Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase.
abstract: NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and/or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a(3)/Cu(B)). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been suggested that two NO molecules can interact with the enzyme simultaneously. We used steady-state and kinetic modeling techniques to reevaluate NO inhibition of CCO. At high flux and low oxygen tensions NO interacts predominantly with the fully reduced (ferrous/cuprous) center in competition with oxygen. However, as the oxygen tension is raised (or the consumption rate is decreased) the reaction with the oxidized enzyme becomes increasingly important. There is no requirement for NO to bind to the singly reduced binuclear center. NO interacts with either ferrous heme iron or oxidized copper, but not both simultaneously. The affinity (K(D)) of NO for the oxygen-binding ferrous heme site is 0.2 nM. The noncompetitive interaction with oxidized copper results in oxidation of NO to nitrite and behaves kinetically as if it had an apparent affinity of 28 nM; at low levels of NO, significant binding to copper can occur without appreciable enzyme inhibition. The combination of competitive (heme) and noncompetitive (copper) modes of binding enables NO to interact with mitochondria across the full in vivo dynamic range of oxygen tension and consumption rates.

title: On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide.
abstract: The detailed molecular mechanism for the reversible inhibition of mitochondrial respiration by NO has puzzled investigators: The rate constants for the binding of NO and O2 to the reduced binuclear center CuB/a3 of cytochrome oxidase (COX) are similar, and NO is able to dissociate slowly from this center whereas O2 is kinetically trapped, which altogether seems to favor the complex of COX with O2 over the complex of COX with NO. Paradoxically, the inhibition of COX by NO is observed at high ratios of O2 to NO (in the 40-500 range) and is very fast (seconds or faster). In this work, we used simple mathematical models to investigate this paradox and other important biological questions concerning the inhibition of COX by NO. The results showed that all known features of the inhibition of COX by NO can be accounted for by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of COX. Besides conciliating apparently contradictory data, this work provided an explanation for the so-called excess capacity of COX by showing that the COX activity found in tissues actually is optimized to avoid an excessive inhibition of mitochondrial respiration by NO, allowing a moderate, but not excessive, overlap between the roles of NO in COX inhibition and in cellular signaling. In pathological situations such as COX-deficiency diseases and chronic inflammation, an excessive inhibition of the mitochondrial respiration is predicted.

title: On the biologic role of the reaction of NO with oxidized cytochrome c oxidase.
abstract: The inhibition of cytochrome c oxidase (CcOX) by nitric oxide (NO) is analyzed with a mathematical model that simulates the metabolism in vivo. The main results were the following: (a) We derived novel equations for the catalysis of CcOX that can be used to predict CcOX inhibition in any tissue for any [NO] or [O(2)]; (b) Competitive inhibition (resulting from the reversible binding of NO to reduced CcOX) emerges has the sole relevant component of CcOX inhibition under state 3 in vivo; (c) In state 4, contribution of uncompetitive inhibition (resulting from the reaction of oxidized CcOX with NO) represents a significant nonmajority fraction of inhibition, being favored by high [O(2)]; and (d) The main biologic role of the reaction between NO and oxidized CcOX is to consume NO. By reducing [NO], this reaction stimulates, rather than inhibits, respiration. Finally, we propose that the biologic role of NO as an inhibitor of CcOX is twofold: in state 4, it avoids an excessive buildup of mitochondrial membrane potential that triggers rapid production of oxidants, and in state 3, increases the efficiency of oxidative phosphorylation by increasing the ADP/O ratio, supporting the therapeutic use of NO in situations in which mitochondria are dysfunctional.

title: The assumption that nitric oxide inhibits mitochondrial ATP synthesis is correct
abstract: The assumption that reversible inhibition of mitochondrial respiration by nitric oxide (NO⋅) represents inhibition of ATP synthesis is unproven. NO⋅ could theoretically inhibit the oxygen consumption with continued ATP synthesis, by acting as an electron acceptor from cytochrome c or as a terminal electron acceptor in stead of oxygen. We report here that NO⋅ does reversibly inhibit brain mitochondrial ATP synthesis with a time course similar to its inhibition of respiration. Whilst such inhibition was largely reversible, there appeared to be a small irreversible component which may theoretically be due to peroxynitrite formation, i.e. as a result of the reaction between NO⋅ and superoxide, generated by the mitochondrial respiratory chain.

title: Glycolysis: a bioenergetic or a survival pathway?
abstract: Following inhibition of mitochondrial respiration neurons die rapidly, whereas astrocytes utilize glycolytically-generated ATP to increase their mitochondrial membrane potential, thus becoming more resistant to pro-apoptotic stimuli. Neurons are unable to increase glycolysis due to the lack of activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, isoform 3 (PFKFB3). In neurons, PFKFB3 is degraded constantly via the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)- CDH1. Glucose metabolism in neurons is directed mainly to the pentose phosphate pathway, leading to regeneration of reduced glutathione. In addition to their relevance to brain physiology and pathophysiology, these observations suggest that APC/C-CDH1 might link activation of glycolysis and cell proliferation as it is also involved in the regulation of cell cycle proteins.

title: Different responses of astrocytes and neurons to nitric oxide: The role of glycolytically generated ATP in astrocyte protection
abstract: It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Δψm) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes—cell types that differ greatly in their glycolytic capacity. Continuous and significant (≈85%) inhibition of respiration by NO (1.4 μM at 175 μM O2) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by ≈25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Δψm and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F1F0-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Δψm and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Δψm and cell death, NO and staurosporin synergized in decreasing Δψm and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell.

title: Nanomolar melatonin enhances nNOS expression and controls HaCaT‐cells bioenergetics
abstract: A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, ∼ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide‐induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.

title: Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells.
abstract: Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.

title: Nitric oxide and AMPK cooperatively regulate PGC‐1α in skeletal muscle cells
abstract: Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator‐activated receptor‐γ coactivator 1α (PGC‐1α). Further, we have shown that nitric oxide interacts with the metabolic sensor enzyme, AMPK. Therefore, we tested the hypothesis that nitric oxide and AMPK act synergistically to upregulate PGC‐1α mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S‐nitroso‐N‐penicillamine (SNAP, 25 μm) or diethylenetriamine‐NONO (DETA‐NO, 50 μm), exhibited elevated AMPK phosphorylation, PGC‐1α mRNA and protein, and basal and uncoupled mitochondrial respiration (P < 0.05). Pre‐treatment of cultures with the AMPK inhibitor, Compound C, prevented these effects. Knockdown of AMPKα1 in L6 myotubes using siRNA reduced AMPKα protein content and prevented upregulation of PGC‐1α mRNA by DETA‐NO. Meanwhile, siRNA knockdown of AMPKα2 had no effect on total AMPKα protein content or PGC‐1α mRNA. These results suggest that NO effects on PGC‐1α expression are mediated by AMPKα1. Paradoxically, we found that the AMPK‐activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR‐induced upregulation of PGC‐1α mRNA was prevented by inhibition of NOS with NG‐nitro‐l‐arginine methyl ester (l‐NAME, 1 mm). Additionally, incubation of isolated mouse extensor digitorum longus (EDL) muscles with 2 mm AICAR for 20 min or electrical stimulation (10 Hz, 13 V) for 10 min induced phosphorylation of AMPKα (P < 0.05), which was completely prevented by pre‐treatment with the NOS inhibitor, l‐NG‐monomethyl arginine (l‐NMMA, 1 mm). These data identify the AMPKα1 isoform as the mediator of NO‐induced effects in skeletal muscle cells. Further, this study supports a proposed model of synergistic interaction between AMPK and NOS that is critical for maintenance of metabolic function in skeletal muscle cells.

title: Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes.
abstract: 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.

title: Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.
abstract: We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

title: TNF-alpha impairs regulation of muscle oxidative phenotype: implications for cachexia?
abstract: Chronic obstructive pulmonary disease (COPD) is characterized by weight loss, muscle wasting (in advanced disease ultimately resulting in cachexia), and loss of muscle oxidative phenotype (oxphen). This study investigates the effect of inflammation (as a determinant of muscle wasting) on muscle oxphen by using cell studies combined with analyses of muscle biopsies of patients with COPD and control participants. We analyzed markers (citrate synthase, β-hydroxyacyl-CoA dehydrogenase, and cytochrome c oxidase IV) and regulators (PGC-1α, PPAR-α, and Tfam) of oxphen in vastus lateralis muscle biopsies of patients with advanced COPD and healthy smoking control participants. Here 17 of 73 patients exhibited elevated muscle TNF-α mRNA levels. In these patients, significantly lower mRNA levels of all oxidative markers/regulators were found. Interestingly, these patients also had a significantly lower body mass index and tended to have less muscle mass. In cultured muscle cells, mitochondrial protein content and myosin heavy chain isoform I (but not II) protein and mRNA levels were reduced on chronic TNF-α stimulation. TNF-α also reduced mitochondrial respiration in a nuclear factor kappaB (NF-κB) -dependent manner. Importantly, TNF-α-induced NF-κB activation decreased promoter transactivation and transcriptional activity of regulators of mitochondrial biogenesis and muscle oxphen. In conclusion, these results demonstrate that TNF-α impairs muscle oxphen in a NF-κB-dependent manner.

title: TNF-alpha reduces PGC-1alpha expression through NF-kappaB and p38 MAPK leading to increased glucose oxidation in a human cardiac cell model.
abstract: Inflammatory responses in the heart that are driven by sustained increases in cytokines have been associated with several pathological processes, including cardiac hypertrophy and heart failure. Emerging data suggest a link between cardiomyopathy and myocardial metabolism dysregulation. To further elucidate the relationship between a pro-inflammatory profile and cardiac metabolism dysregulation, a human cell line of cardiac origin, AC16, was treated with tumour necrosis factor-alpha (TNF-alpha).



Exposure of AC16 cells to TNF-alpha inhibited the expression of peroxisome proliferator-activated receptor coactivator 1alpha (PGC-1alpha), an upstream regulator of lipid and glucose oxidative metabolism. Studies performed with cardiac-specific transgenic mice (Mus musculus) overexpressing TNF-alpha, which have been well characterized as a model of cytokine-induced cardiomyopathy, also displayed reduced PGC-1alpha expression in the heart compared with that of control mice. The mechanism by which TNF-alpha reduced PGC-1alpha expression in vitro appeared to be largely mediated via both p38 mitogen-activated protein kinase and nuclear factor-kappaB pathways. PGC-1alpha downregulation resulted in an increase in glucose oxidation rate, which involved a reduction in pyruvate dehydrogenase kinase 4 expression and depended on the DNA-binding activity of both peroxisome proliferator-activated receptor beta/delta and estrogen-related receptor alpha transcription factors.



These results point to PGC-1alpha downregulation as a potential contributor to cardiac dysfunction and heart failure in metabolic disorders with an inflammatory background.

title: Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly.
abstract: Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.

title: Diffusion of peroxynitrite across erythrocyte membranes.
abstract: Peroxynitrite anion (ONOO-) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. At present, however, there is limited knowledge about the mechanisms of peroxynitrite diffusion through biological compartments. In this work we have studied the diffusion of peroxynitrite across erythrocyte membranes. In solution, peroxynitrite rapidly reacts with oxyhemoglobin to yield methemoglobin, with k2 = (10.4 +/- 0.3) x 10(3) M-1.s-1 at pH 7.4 and 25 degrees C. Addition of peroxynitrite to intact erythrocytes caused oxidation of intracellular oxyhemoglobin to methemoglobin. Oxidation yields in red blood cells at pH 7.0 were approximately 40% of those obtained in solution, which results mostly from competition of other cytosolic components for peroxynitrite. Indeed, rather small differences were observed between oxidation yields in lysates compared with intact erythrocytes, in particular at acidic and neutral pH values, indicating that membrane was not precluding peroxynitrite diffusion. Incubation of erythrocytes at pH 7.0 with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a specific inhibitor of anion exchange, resulted in up to 50% inhibition of oxyhemoglobin oxidation by peroxynitrite. More protection by DIDS was achieved at alkaline pH, while no effect was observed at pH 5.5, where 95% of peroxynitrite is in the acidic form, ONOOH (pKa = 6.8). In addition, peroxynitrite caused nitration of intracellular hemoglobin, in a process that was enhanced in thiol-depleted erythrocytes. Our results indicate that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the DIDS-inhibitable anion channel, and in the protonated form by passive diffusion.

title: Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.
abstract: Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.

title: Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite.
abstract: Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.

title: Biochemical properties of cytochrome c nitrated by peroxynitrite.
abstract: Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.

title: Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite.
abstract: Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.

title: Inhibition of mitochondrial electron transport by peroxynitrite.
abstract: Mammalian mitochondria are sensitive targets of the cytotoxic effects of superoxide (O.2-) and nitric oxide (.NO). In turn, when superoxide and nitric oxide are simultaneously produced, they rapidly react with each other yielding the highly oxidizing peroxynitrite anion (ONOO-) which may be also toxic to mammalian mitochondria. In this study we report that peroxynitrite exposure to rat heart mitochondria resulted in significant inactivation of electron carriers such as succinate dehydrogenase and NADH dehydrogenase as well as the mitochondrial ATPase. As a result of enzyme inactivation, peroxynitrite lead to a profound inhibition of glutamate/malate- and succinate-supported oxygen consumption but did not cause mitochondrial uncoupling. Secondary to inhibiting mitochondrial electron transport, peroxynitrite induced an enhanced succinate-stimulated hydrogen peroxide formation by heart mitochondria. Most of the damaging effects against mitochondria can be ascribed to peroxynitrite anion itself and not to hydroxyl radical-like oxidant yielded during the proton-catalyzed decomposition of peroxynitrite, as hydroxyl radical scavengers provided a rather modest protection. Our observations indicate that mitochondria may constitute a key intracellular loci for the toxic effects of peroxynitrite under the various pathological conditions in which peroxynitrite appears to play a contributory role.

title: Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation.
abstract: This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

title: Peroxynitrite-Mediated Mitochondrial Dysfunction
abstract: Peroxynitrite anion (ONOO–) is a potent biological oxidant produced by the near diffusion-limited reaction of superoxide and nitric oxide. Peroxynitrite has been implicated in diverse forms of free radical-induced tissue injury. Experimental evidence showed that exogenous and endogenous peroxynitrite causes alterations of the structure and function of mitochondrial proteins, leading to mitochondrial dysfunction and cellular or organ injury. These data are discussed along with its physiopathological implications.

title: Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.
abstract: Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.

title: Peroxynitrite-derived carbonate and nitrogen dioxide radicals readily react with lipoic and dihydrolipoic acid.
abstract: Alpha-lipoic acid (LA) and dihydrolipoic acid (DHLA) may have a role as antioxidants against nitric oxide-derived oxidants. We previously reported that peroxynitrite reacts with LA and DHLA with second-order rate constants of 1400 and 500 M(-1) s(-1), respectively, but indicated that these direct reactions are not fast enough to protect against peroxynitrite-mediated damage in vivo. Moreover, the mechanism of the reaction of peroxynitrite with LA has been recently challenged ( 2004). Pulse radiolysis studies indicate that LA and DHLA react with peroxynitrite-derived nitrogen dioxide (*NO2) (k2 = 1.3 x 10(6) and 2.9 x 10(7) M(-1) s(-1), respectively) and carbonate radicals (CO(3-)) (k2 = 1.6 x 10(9) and 1.7 x 10(8) M(-1) s(-1), respectively). Carbonate radical-mediated oxidation of LA led to the formation of the potent one-electron oxidant LA radical cation. LA inhibited peroxynitrite-mediated nitration of tyrosine and of a hydrophobic tyrosine analog, N-t-BOC L-tyrosine tert-butyl ester (BTBE), incorporated into liposomes but enhanced tyrosine dimerization. Moreover, while LA competitively inhibited the direct oxidation of glutathione by peroxynitrite, it was poorly effective against the radical-mediated thiol oxidation. The mechanisms of reaction defined herein allow to rationalize the biochemistry of peroxynitrite based on direct and free radical-mediated processes and contribute to the understanding of the antioxidant actions of LA and DHLA.

title: Effect of L‐buthionine‐(S,R)‐sulphoximine, an inhibitor of γ‐glutamylcysteine synthetase on peroxynitrite‐ and endotoxic shock‐induced vascular failure
abstract: Peroxynitrite, a cytotoxic oxidant formed from the reaction of nitric oxide (NO) and superoxide is a mediator of cellular injury in ischaemia/reperfusion injury, shock and inflammation. Here we investigated whether L‐buthionine‐(S,R)‐sulphoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase, alters endothelial and vascular smooth muscle injury in response to peroxynitrite in vitro and during endotoxic shock in vivo. In human umbilical vein endothelial cells and in rat aortic smooth muscle cells, BSO (1 mM, for 24 h) enhanced, whereas glutathione (3 mM) or glutathione ethyl ester (3 mM) attenuated the peroxynitrite (100–1000 μM)‐induced suppression of mitochondrial respiration (measured by the conversion of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) to formazan), formation of nitrotyrosine (detected by Western blotting), protein oxidation (measured by detection of 2,4 dinitrophenylhydrazine‐reactive carbonyls), and DNA single strand breakage and activation of the nuclear enzyme poly (ADP‐ribose) synthetase (PARS) (measured by the incorporation of radiolabelled NAD+ into nuclear proteins and by the alkaline unwinding assay, respectively). Glutathione ethyl ester treatment reduced the BSO‐induced enhancement of peroxynitrite‐induced cytotoxicity. In rat isolated thoracic aortic rings, BSO treatment (in vivo, at 1 g kg−1 intraperitoneally (i.p.) for 24 h) enhanced, whereas pretreatment with glutathione (in vitro, 3 mM) attenuated the peroxynitrite‐induced reduction of the contractions to noradrenaline, and the peroxynitrite‐induced impairment of the endothelium‐dependent relaxations to acetylcholine. In BSO‐pretreated rats, treatment with bacterial lipopolysaccharide (LPS, 15 mg kg−1, i.p., for 6 h) caused a more pronounced vascular hyporeactivity and endothelial dysfunction ex vivo. BSO pretreatment also increased the degree of nitrotyrosine staining (detected by imunohistochemistry) in the aorta after LPS treatment. In conclusion, our results demonstrate that L‐buthionine‐(S,R)‐sulphoximine, an inhibitor of γ‐glutamylcysteine synthetase enhances peroxynitrite‐ and endotoxic shock‐induced vascular failure. Based on these findings, we suggest that endogenous glutathione plays an important protective role against peroxynitrite‐ and LPS‐induced vascular injury.

title: Protection against peroxynitrite
abstract: Peroxynitrite formed in vivo from superoxide and nitric oxide can mediate oxidation, nitration, or nitrosation reactions, leading to impaired function, toxicity, and alterations in signaling pathways. Protection against peroxynitrite is important for defense of normal tissue, especially during inflammation. Biological protection against peroxynitrite is organized in three categories: prevention, interception, and repair. Prevention is the control of the formation of peroxynitrite precursors, nitric oxide and superoxide. Interception is by direct reaction with peroxynitrite, leading to non‐toxic products. In this regard, organoselenium compounds, metalloporphyrin derivatives, and peroxidases (e.g. glutathione peroxidase and myeloperoxidase) exhibit high second‐order rate constants with peroxynitrite. Ebselen, like glutathione peroxidase, protects in a catalytic fashion utilizing glutathione as reductant in the peroxynitrite reductase reaction. Protection by metalloporphyrins can be maintained through glutathione or ascorbate. Repair processes remove damaged products and restitute intact biomolecules.

title: Protective effect of N-acetylcysteine on multiple organ failure induced by zymosan in the rat.
abstract: In the present study, we evaluated the effect of N-acetylcysteine treatment in a nonseptic shock model induced by zymosan in the rat. Animals were randomly divided into eight groups (ten animals in each group). The first group was treated with ip administration of saline solution (0.90% NaCl) and served as the sham group. The second group was treated with ip administration of zymosan (500 mg/kg suspended in saline solution). In the third and fourth groups, rats received ip administration of N-acetylcysteine (40 mg/kg; 1 and 6 hrs after administration of zymosan or saline). In the fifth and sixth groups, rats received ip administration of N-acetylcysteine (20 mg/kg; 1 and 6 hrs after zymosan or saline administration). In the seventh and eighth groups, rats received ip administration of N-acetylcysteine (10 mg/kg; 1 and 6 hrs after zymosan or saline administration). After zymosan or saline injection, animals were monitored for the evaluation of systemic toxicity (conjunctivitis, ruffled fur, diarrhea, and lethargy), loss of body weight, and mortality for 72 hrs. Exudate formation, leukocyte infiltration, nitrate/nitrite production, lung and intestine myeloperoxidase activity and lipid peroxidation, and histologic examination were evaluated at 18 hrs after zymosan administration.



Administration of zymosan in the rat induced acute peritonitis, as assessed by a marked increase in the leukocyte count in the exudate, as well as by an increase in the exudate nitrate/nitrite concentration. Lung and intestine myeloperoxidase activity and lipid peroxidation was significantly increased in zymosan-treated rats. This inflammatory process coincided with the damage of lung and small intestine. Peritoneal administration of zymosan in the rat also induced a significant increase in the plasma levels of nitrite and nitrate and stable metabolites of nitric oxide and in levels of peroxynitrite, as measured by the oxidation of the fluorescent dihydrorhodamine 123 at 18 hrs after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the lung of zymosan-shocked rats. Pretreatment of zymosan-shocked rats with ip administration of N-acetylcysteine (40, 20, and 10 mg/kg, 1 and 6 hrs after zymosan) prevented the development of peritonitis and reduced peroxynitrite formation in a dose-dependent manner. In addition, ip administration of N-acetylcysteine (40 mg/kg, 1 and 6 hrs after zymosan) was effective in preventing the development of lung and intestine injury and neutrophil infiltration, as determined by myeloperoxidase evaluation.



Taken together, the present results demonstrate that N-acetylcysteine exerts potent anti-inflammatory effects.

title: Modifications of Glyceraldehyde-3-Phosphate Dehydrogenase Induced by Increasing Concentrations of Peroxynitrite: Early Recognition by 20S Proteasome
abstract: Peroxynitrite, a potent oxidizing and nitrating species, induces covalent modifications of biomolecules in a number of pathological conditions. In previous studies with S. cerevisiae, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as being especially susceptible to nitration by peroxynitrite. The activity of this enzyme was strongly inhibited by low doses of peroxynitrite in yeast and in cultured rat astrocytes. Here, the sequence of modifications of isolated mammalian GAPDH induced by increasing concentrations of peroxynitrite is demonstrated to be as follows: (i) oxidation, leading to inactivation and to enhanced susceptibility of GAPDH for proteasomal degradation, (ii) oligomer formation, and (iii) nitration. In our study the susceptibility for degradation by isolated 20S proteasome was by far the most sensitive parameter for peroxynitrite-induced damage to GAPDH, implying that this might also occur under pathological conditions where peroxynitrite is generated at low concentrations in vivo.

title: Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite.
abstract: Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inactivated by peroxynitrite under biologically relevant conditions. The decrease of enzymatic activity followed an exponential function, and the concentration of peroxynitrite needed to inactivate 50% of 7 microM GAPDH (IC50) was 17 microM. Hydroxyl radical scavengers did not protect GAPDH from inactivation, but molecules that react directly with peroxynitrite such as cysteine, glutathione, or methionine and the substrate, glyceraldehyde 3-phosphate, afforded significant protection. Assuming simple competition kinetics between scavengers and the enzyme, we estimated a second-order rate constant of (2.5 +/- 0.5) x 10(5) M-1 s-1 at 25 degreesC and pH 7.4 for the GAPDH tetramer. The loss of enzyme activity was accompanied by protein thiol oxidation (two thiols oxidized per subunit) with only one critical thiol responsible of enzyme inactivation. Indeed, the pH profile of inactivation was consistent with the reaction of GAPDH sulfhydryls (GAPDH-SH) with peroxynitrite. Peroxynitrite-inactivated GAPDH was resistant to arsenite reduction and only 15% recovered by 20 mM dithiothreitol, suggesting that GAPDH-SH has been mainly oxidized to sulfinic or sulfonic acid, with a minor proportion yielding a disulfide. On the other hand, under anaerobic conditions the peroxynitrite precursor, nitric oxide (*NO), only slowly inactivated GAPDH with a rate constant of 11 M-1 s-1. The remarkable reactivity of the critical thiol group in GAPDH (Cys-149) toward peroxynitrite, which is one order of magnitude higher than that of previously studied sulfhydryls, indicate that it may constitute a preferential intracellular target for peroxynitrite.

title: Rapid and irreversible inhibition of creatine kinase by peroxynitrite
abstract: We examined the ability of peroxynitrite and other ⋅NO‐derived oxidants to inhibit creatine kinase (CK). Peroxynitrite potently inhibited CK activity and depleted protein thiols. The rate constant for this reaction was 8.85×105 M−1 s−1. Glutathione did not reactivate CK activity nor did it regenerate protein thiol content. In contrast, glutathione reactivated CK, and regenerated protein thiols, after inhibition by either ⋅NO or oxidized glutathione (GSSG). Peroxynitrite did not irreversibly inhibit CK after it had been treated with GSSG to block protein thiols. We conclude that thiol oxidation is a critical event leading to inactivation of CK by peroxynitrite.

title: Effect of Peroxynitrite on the Mitochondrial Respiratory Chain: Differential Susceptibility of Neurones and Astrocytes in Primary Culture
abstract: The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO−), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO− (initial concentrations of 0.01–2.0 mM) caused, after a subsequent 24‐h incubation, a dose‐dependent decrease in succinate‐cytochrome c reductase (60% at 0.5 mM) and in cytochrome c oxidase (52% at 0.5 mM) activities. NADH‐ubiquinone‐1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 mM ONOO−. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO− in a dose‐dependent manner (40% at 0.5 mM ONOO−) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 mM ONOO−, but astrocytic glutathione was not affected up to 2 mM ONOO−. In isolated brain mitochondria, only succinate‐cytochrome c reductase activity was affected (22% decrease at 1 mM ONOO−). We conclude that the acute exposure of ONOO− selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO−‐mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.

title: Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors.
abstract: The effects of both nitric oxide (NO) and peroxynitrite on complexes I (NADH dehydrogenase) and III (cytochrome c reductase) isolated from bovine heart have been examined. EPR signals ("g=2.01") previously detected in association with loss of complex I and III activities in cultured cells and isolated mitochondria subjected to nitrosative stress are shown not to arise from these particular enzymes. Neither NO nor peroxynitrite (ONO(2)(-)) reacts to any appreciable extent with the oxidized forms of flavin mononucleotide, iron-sulfur clusters, or heme moieties found in complexes I and III. However, ONO(2)(-) is readily able to abstract electrons from the reduced forms of both complexes I and III, without any apparent modification of the enzyme cofactors. While no attempt was made in the present study to catalog all the possible modifications, it is clear that ONO(2)(-) can react with the protein moieties of the enzymes. For example, when added in excess, ONO(2)(-) derivatizes a select few tyrosine residues in both complexes I and III forming 3-nitrotyrosine as detected by immunoblots. In the case of complex I, we find a minimum of 3 out of the 46 subunits present were modified (49, approximately 18, and approximately 15kDa); whereas in complex III, 4 out of the 13 subunits stained for 3-nitrotyrosine (46, 27, 7, and 6kDa). Significant irreversible inhibition of activity required the addition of >10(2)-fold excesses of ONO(2)(-) to the enzymes. At 10(3)-fold excess of added ONO(2)(-), the activity of complex I was only diminished by approximately 18%, while a 60% loss of activity was observed for complex III.

title: Nitric Oxide and Peroxynitrite Interactions with Mitochondria
abstract: Nitric oxide (NO) and peroxynitrite (ONOO) avidly interact with mitochondrial components, leading to a range of biological responses spanning from the modulation of mitochondrial respiration, mitochondrial dysfunction to the signaling of apoptotic cell death. Physiological levels of NO primarily interact with cytochrome c oxidase, leading to a competitive and reversible inhibition of mitochondrial oxygen uptake. In turn, this leads to alterations in electrochemical gradients, which affect calcium uptake and may regulate processes such as mitochondrial transition pore (MTP) opening and the release of proapoptotic proteins. Large or persistent levels of NO in mitochondria promote mitochondrial oxidant formation. Peroxynitrite formed either extra or intramitochondrially leads to oxidative damage, most notably at complexes I and II of the electron transport chain, ATPase, aconitase and Mnsuperoxide dismutase. Mitochondrial scavenging systems for peroxynitrite and peroxynitritederived radicals such as carbonate (CO3.) and nitrogen dioxide radicals (NO2) include cytochrome c oxidase, glutathione and ubiquinol and serve to partially attenuate the reactions of these oxidants with critical mitochondrial targets. Detection of nitrated mitochondrial proteins in vivo supports the concept that mitochondria constitute central loci of the toxic effects of excess reactive nitrogen species. In this review we will provide an overview of the biochemical mechanisms by which NO and ONOO regulate or alter mitochondrial functions.

title: Peroxynitrite reactions and formation in mitochondria.
abstract: Mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, and these interactions are recognized to contribute to the biological and pathological effects of both nitric oxide ((*)NO) and peroxynitrite. Extra- or intramitochondrially formed peroxynitrite can diffuse through mitochondrial compartments and undergo fast direct and free radical-dependent target molecule reactions. These processes result in oxidation, nitration, and nitrosation of critical components in the matrix, inner and outer membrane, and intermembrane space. Mitochondrial scavenging and repair systems for peroxynitrite-dependent oxidative modifications operate but they can be overwhelmed under enhanced cellular (*)NO formation as well as under conditions that lead to augmented superoxide formation by the electron transport chain. Peroxynitrite can lead to alterations in mitochondrial energy and calcium homeostasis and promote the opening of the permeability transition pore. The effects of peroxynitrite in mitochondrial physiology can be largely rationalized based on the reactivities of peroxynitrite and peroxynitrite-derived carbonate, nitrogen dioxide, and hydroxyl radicals with critical protein amino acids and transition metal centers of key mitochondrial proteins. In this review we analyze (i) the existing evidence for the intramitochondrial formation and reactions of peroxynitrite, (ii) the key reactions and fate of peroxynitrite in mitochondria, and (iii) their impact in mitochondrial physiology and signaling of cell death.

title: Cytochrome c Nitration by Peroxynitrite*
abstract: Peroxynitrite (ONOO−), the product of superoxide (O⨪2) and nitric oxide (·NO) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO− during conditions involving accelerated rates of oxygen radical and ·NO generation. Horse heart cytochromec 3+ was nitrated by ONOO−, as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochromec 3+ 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO2) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochromec 3+ was more extensively nitrated than cytochrome c 2+ because of the preferential oxidation of the reduced heme by ONOO−. Similar protein nitration patterns were obtained by ONOO− reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO2 −) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during ·NO- and ONOO−-mediated cell injury.

title: Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione.
abstract: Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.

title: Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide.
abstract: Mitochondrial and cytosolic aconitases have been indicated as major targets of .NO- and O2-.-mediated toxicity in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. However, under circumstances in which both .NO and O2-. are generated, their almost diffusion-controlled combination reaction (k = 6.7 x 10(9) M-1 s-1), leading to the formation of peroxynitrite anion (ONOO-), can out-compete the direct reactions of .NO and O2-. with aconitase and even the enzymatic dismutation of O2-. by superoxide dismutase. In this work, we report that ONOO- reacts with isolated pig heart mitochondrial aconitase at 1.4 x 10(5) M-1 s-1, resulting in a significant loss of enzymatic activity. Aconitase activity was totally recovered after postincubation with thiols and ferrous iron, indicating that ONOO- reactions with the enzyme involve the perturbation of the labile Fe alpha to yield the inactive [3Fe-4S] cluster, which is also evident by spectral changes. On the other hand, anaerobic exposure of isolated aconitase to high concentrations of .NO (> 100 microM) led to a moderate inhibition of the enzyme, which could be fully overcome by .NO displacement under an argon-saturated atmosphere, in agreement with the formation of a reversible inhibitory complex between .NO and the active site of aconitase. Superoxide inactivated mitochondrial aconitase at (3.5 +/- 2) x 10(6) M-1 s-1, a reaction rate 3 orders of magnitude slower than its reaction rate with .NO. O2-. could represent the main mechanism of inactivation of the enzyme in systems in which it is formed without significant concomitant production of .NO. Our results imply that the mechanisms by which .NO and O2-. inactivate aconitase in cell systems may not be simple due to their direct reactions with the iron-sulfur cluster, but may rely on the formation of ONOO-.

title: Oxidative modification of nicotinamide nucleotide transhydrogenase in submitochondrial particles: effect of endogenous ubiquinol.
abstract: The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.

title: The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal
abstract: Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

title: Alterations to glutathione and nicotinamide nucleotides during the mitochondrial permeability transition induced by peroxynitrite.
abstract: Peroxynitrite is a biologically important oxidant that damages mitochondria in a number of ways. We investigated the interaction of peroxynitrite with the mitochondrial glutathione pool by measuring the formation of oxidised glutathione and glutathione-protein mixed disulfides in mitochondria exposed to either peroxynitrite or tert-butylhydroperoxide. In contrast to tert-butylhydroperoxide, peroxynitrite converts 40-50% of mitochondrial glutathione to products other than disulfides, primarily higher oxidation states of sulfur. These data show that peroxynitrite interacts with mitochondria quite differently from oxidants commonly used in studying mitochondrial oxidative stress. Peroxynitrite also induces a permeability transition in the mitochondrial inner membrane, and here we show that this permeability transition is prevented by the NAD(P)H-linked substrates glutamate and malate and by the thiol reagent dithiothreitol. Glutamate and malate prevented complete oxidation of the NAD(P)H pool by peroxynitrite or tert-butylhydroperoxide but did not prevent oxidation of the mitochondrial glutathione pool or the formation of glutathione-protein mixed disulfides. This study is consistent with regulation of the permeability transition by critical protein thiol groups, whose redox state responds to that of the mitochondrial NAD(P)H pool, but which do not equilibrate directly with the mitochondrial glutathione pool.

title: Protective Role of Malvidin-3-Glucoside on Peroxynitrite-Induced Damage in Endothelial Cells by Counteracting Reactive Species Formation and Apoptotic Mitochondrial Pathway
abstract: The health-promoted benefits of anthocyanins, including vascular protective effects and antiatherogenic properties, have now been recognized, but the involved molecular mechanisms have not been well elucidated. Following our previous work on cytoprotective mechanisms of some anthocyanins against apoptosis triggered by peroxynitrite in endothelial cells, here we investigated the protective role of malvidin-3-glucoside, a major dietary anthocyanin, on such deleterious process, by exploring the interference on cellular reactive species formation and on apoptotic mitochondrial pathway. Preincubation of cells with 25 μM malvidin-3-glucoside protected efficiently endothelial cells from peroxynitrite-promoted apoptotic death, an effect which may be partially mediated by its ability to decrease the formation of reactive species after cell aggression, as assessed by the dichlorodihydrofluorescein diacetate assay and by carbonyl groups formation. Moreover, malvidin-3-glucoside inhibited mitochondrial apoptotic signaling pathways induced by peroxynitrite, by counteracting mitochondrial membrane depolarization, the activation of caspase-3 and -9, and the increase in the expression of the proapoptotic Bax protein. Altogether, our data expands our knowledge about the molecular mechanisms underlying the vascular protection afforded by malvidin-3-glucoside, and anthocyanins in general, in the context of prevention of endothelial dysfunction and atherosclerosis.

title: Interaction of Peroxynitrite with Mitochondrial Cytochrome Oxidase
abstract: Purified mitochondrial cytochrome coxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear hemea 3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a 3nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreasedV max and a raised K m for oxygen. Under these conditions, the redox state of cytochromea is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 μm peroxynitrite to 10 μm fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochromea 3, partial reduction of cytochromea, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 μm) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a anda 3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.

title: DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.
abstract: The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.

title: Nitric oxide and peroxynitrite in health and disease.
abstract: The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

title: Oxidative stress and regional ischemia-reperfusion injury: the peroxynitrite–poly(ADP-ribose) polymerase connection
abstract: Inotek Pharmaceuticals Corporation, Beverly, Massachusetts, USA, Department of Internal Medicine, University Hospital, Lausanne, Switzerland, Department of Cardiac Surgery, University of Heidelberg, Germany and Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary.  This study was supported by a grant from the National Institutes of Health (R01HL59266) to C.S.L.L. was supported by a grant from the ADUMED Foundation (Switzerland).

title: Multiple pathways of peroxynitrite cytotoxicity.
abstract: Peroxynitrite is a reactive oxidant produced from nitric oxide (NO) and superoxide, which reacts with a variety of biomolecules including proteins, lipids and DNA. Peroxynitrite is produced by the body in response to a variety of toxicologically relevant molecules including environmental toxins. It is also produced by the body in response to environmental toxins, as well as in reperfusion injury and inflammation. Here we overview the multiple pathways of peroxynitrite cytotoxicity. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na(+)/K(+) ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of peroxynitrite. In addition, peroxynitrite is a potent trigger of DNA strand breakage, with subsequent activation of the nuclear enzyme poly-ADP ribosyl synthetase or polymerase (PARP), with eventual severe energy depletion and necrosis of the cells. Studies conducted with peroxynitrite decomposition catalysts suggest that neutralization of peroxynitrite is of significant therapeutic benefit after exposure to various environmental toxins as well as in a variety of inflammatory and reperfusion disease conditions.

title: Poly(adenosine 5′-diphosphate) ribose polymerase activation as a cause of metabolic dysfunction in critical illness
abstract: Poly(adenosine 5′-diphosphate) ribose polymerase is a nuclear enzyme activated in response to genotoxic stress induced by a variety of DNA damaging agents. Several oxygen and nitrogen-centered free radicals, notably peroxynitrite, are strong inducers of DNA damage and poly(adenosine 5′-diphosphate) ribose polymerase activation in vitro and in vivo. Activation of this nuclear enzyme depletes the intracellular stores of its substrate nicotinamide adenine dinucleotide, slowing the rate of glycolysis, mitochondrial electron transport and adenosine triphosphate formation. This process triggers a severe energetic crisis within the cell, leading to acute cell dysfunction and cell necrosis. Poly(adenosine 5′-diphosphate) ribose polymerase also plays an important role in the regulation of inflammatory cascades, through a functional association with various transcription factors and transcription co-activators. Recent works identified this enzyme as a critical mediator of cellular metabolic dysfunction, inflammatory injury, and organ damage in conditions associated with overwhelming oxidative stress, including systemic inflammation, circulatory shock, and ischemia-reperfusion. Accordingly, pharmacological inhibitors of poly(adenosine 5′-diphosphate) ribose polymerase protect against cell death and tissue injury in such conditions, and may therefore represent novel therapeutic tools to limit multiple organ damage and dysfunction in critically ill patients.

title: Role of poly(adenosine diphosphate–ribose) polymerase 1 in septic peritonitis
abstract: Peritonitis generally results from gastrointestinal perforation, with systemic sepsis developing over hours or days from an initially localized nidus of infection. The consecutive inflammatory response induces the widespread generation of oxidants and free radicals, which are potent inducers of breaks and nicks in double-stranded DNA. This genetic damage triggers the activation of the nuclear enzyme poly(ADP-ribose) polymerase 1, which, in turn, cleaves the respiratory coenzyme nicotinamide adenine dinucleotide into nicotinamide and ADP ribose. The consecutive decrease in cellular nicotinamide adenine dinucleotide inhibits glycolysis and mitochondrial respiration, leading to cellular energy collapse and necrotic cell death. In parallel, poly(ADP-ribose) polymerase 1 positively regulates inflammatory signal transduction pathways through a functional association with the transcription factor nuclear factor &kgr;B, resulting in a progressive amplification of local inflammation. Recent data indicate that these molecular mechanisms are instrumental in the development of cardiovascular collapse and multiple organ dysfunction in sepsis, supporting the view that pharmacologic inhibitors of poly(ADP-ribose) polymerase 1 may represent useful tools for the treatment of this condition.

title: Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion.
abstract: Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate beta-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP(-/-)) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP(-/-) animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis.

title: Redox modulation of p53 conformation and sequence-specific DNA binding in vitro.
abstract: The p53 protein is a transcription factor, the function of which is abrogated by oncogenic mutations which affect a flexible domain in the central portion of p53, altering its reactivity with conformation-specific antibodies. Here we show that both conformation and sequence-specific DNA binding of p53 translated in vitro can be modulated by metal chelators and oxidizing agents. Oxidation disrupted wild-type p53 conformation and inhibited DNA binding. Conversely, reduction favored folding of p53 into the wild-type form and restored DNA binding. Redox regulation of p53 protein conformation could represent an important mechanism for the control of p53 function.

title: Conformational Altered p53 as an Early Marker of Oxidative Stress in Alzheimer's Disease
abstract: In order to study oxidative stress in peripheral cells of Alzheimer's disease (AD) patients, immortalized lymphocytes derived from two peculiar cohorts of patients, referring to early onset AD (EOSAD) and subjects harboured AD related mutation (ADmut), were used. Oxidative stress was evaluated measuring i) the typical oxidative markers, such as HNE Michel adducts, 3 Nitro-Tyrosine residues and protein carbonyl on protein extracts, ii) and the antioxidant capacity, following the enzymatic kinetic of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRD). We found that the signs of oxidative stress, measured as oxidative marker levels, were evident only in ADmut but not in EOSAD patients. However, oxidative imbalance in EOSAD as well as ADmut lymphocytes was underlined by a reduced SOD activity and GRD activity in both pathological groups in comparison with cells derived from healthy subjects. Furthermore, a redox modulated p53 protein was found conformational altered in both EOSAD and ADmut B lymphocytes in comparison with control cells. This conformational altered p53 isoform, named “unfolded p53”, was recognized by the use of two specific conformational anti-p53 antibodies. Immunoprecipitation experiments, performed with the monoclonal antibodies PAb1620 (that recognizes p53wt) and PAb240 (that is direct towards unfolded p53), and followed by the immunoblotting with anti-4-hydroxynonenal (HNE) and anti- 3-nitrotyrosine (3NT) antibodies, showed a preferential increase of nitrated tyrosine residues in unfolded p53 isoform comparing to p53 wt protein, in both ADmut and EOSAD. In addition, a correlation between unfolded p53 and SOD activity was further found. Thus this study suggests that ROS/RNS contributed to change of p53 tertiary structure and that unfolded p53 can be considered as an early marker of oxidative imbalance in these patients.

title: Conformational altered p53 affects neuronal function: relevance for the response to toxic insult and growth-associated protein 43 expression
abstract: The role of p53 in neurodegenerative diseases is essentially associated with neuronal death. Recently an alternative point of view is emerging, as altered p53 conformation and impaired protein function have been found in fibroblasts and blood cells derived from Alzheimer’s disease patients. Here, using stable transfected SH-SY5Y cells overexpressing APP751wt (SY5Y-APP) we demonstrated that the expression of an unfolded p53 conformation compromised neuronal functionality. In particular, these cells showed (i) augmented expression of amyloid precursor protein (APP) and its metabolites, including the C-terminal fragments C99 and C83 and β-amyloid peptide (ii) high levels of oxidative markers, such as 4-hydroxy-2-nonenal Michael-adducts and 3-nitro-tyrosine and (iii) altered p53 conformation, mainly due to nitration of its tyrosine residues. The consequences of high-unfolded p53 expression resulted in loss of p53 pro-apoptotic activity, and reduction of growth-associated protein 43 (GAP-43) mRNA and protein levels. The role of unfolded p53 in cell death resistance and lack of GAP-43 transcription was demonstrated by ZnCl2 treatment. Zinc supplementation reverted p53 wild-type tertiary structure, increased cells sensitivity to acute cytotoxic injury and GAP-43 levels in SY5Y-APP clone.

title: Inactivation of wild-type p53 protein function by reactive oxygen and nitrogen species in malignant glioma cells.
abstract: Malignant gliomas are the most common primary brain tumors in adults, and the most malignant form, glioblastoma multiforme (GBM), is usually rapidly fatal. Most GBMs do not have p53 mutations, although the p53 tumor suppressor pathway appears to be inactivated. GBMs grow in a hypoxic and inflammatory microenvironment, and increased levels of the free radicals nitric oxide (NO) and superoxide () occur in these malignancies in vivo. Peroxynitrite (ONOO(-)) is a highly reactive molecule produced by excess NO and that can posttranslationally modify and inactivate proteins, especially zinc finger transcription factors such as p53. We demonstrated previously that GBMs have evidence of tyrosine nitration, the "footprint" of peroxynitrite-mediated protein modification in vivo, and that peroxynitrite could inhibit the specific DNA binding ability of wild-type p53 protein in glioma cells in vitro. Here we show that both authentic peroxynitrite and SIN-1 (3-morpholinosydnonimine hydrochloride), a molecule that decomposes into NO and to form peroxynitrite, can inhibit wild-type p53 function in malignant glioma cells. Concentrations of peroxynitrite associated with a tumor inflammatory environment caused dysregulation of wild-type p53 transcriptional activity and downstream p21(WAF1) expression.

title: Homeostatic functions of the p53 tumor suppressor: regulation of energy metabolism and antioxidant defense.
abstract: The p53 tumor suppressor plays pivotal role in the organism by supervising strict compliance of individual cells to needs of the whole organisms. It has been widely accepted that p53 acts in response to stresses and abnormalities in cell physiology by mobilizing the repair processes or by removing the diseased cells through initiating the cell death programs. Recent studies, however, indicate that even under normal physiological conditions certain activities of p53 participate in homeostatic regulation of metabolic processes and that these activities are important for prevention of cancer. These novel functions of p53 help to align metabolic processes with the proliferation and energy status, to maintain optimal mode of glucose metabolism and to boost the energy efficient mitochondrial respiration in response to ATP deficiency. Additional activities of p53 in non-stressed cells tune up the antioxidant defense mechanisms reducing the probability of mutations caused by DNA oxidation under conditions of daily stresses. The deficiency in the p53-mediated regulation of glycolysis and mitochondrial respiration greatly accounts for the deficient respiration of the predominance of aerobic glycolysis in cancer cells (the Warburg effect), while the deficiency in the p53-modulated antioxidant defense mechanisms contributes to mutagenesis and additionally boosts the carcinogenesis process.

title: A pivotal role for p53: balancing aerobic respiration and glycolysis
abstract: The genetic basis of increased glycolytic activity observed in cancer cells is likely to be the result of complex interactions of multiple regulatory pathways. Here we review the recent evidence of a simple genetic mechanism by which tumor suppressor p53 regulates mitochondrial respiration with secondary changes in glycolysis that are reminiscent of the Warburg effect. The biological significance of this regulation of the two major pathways of energy generation by p53 remains to be seen.

title: p53: new roles in metabolism.
abstract: Virtually all cancers show metabolic changes that result in upregulation of glycolysis and glucose consumption. Although discovered in the 1920s, how this glycolytic switch happens, and whether it is a cause or a consequence of the malignant process, has remained a matter of debate. The p53 tumor suppressor gene, discovered some 30 years ago, has a key role in preventing cancer development. Recent discoveries revealing new functions for p53 in the regulation of glucose metabolism and oxidative stress have brought together these two venerable fields of cancer biology. These activities of p53 appear to be key in tumor suppression, and shed some light on the pathways that underlie the metabolic changes in cancer cells.

title: NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration
abstract: Cell proliferation is a metabolically demanding process. It requires active reprogramming of cellular bioenergetic pathways towards glucose metabolism to support anabolic growth. NF-κB/Rel transcription factors coordinate many of the signals that drive proliferation during immunity, inflammation and oncogenesis, but whether NF-κB regulates the metabolic reprogramming required for cell division during these processes is unknown. Here, we report that NF-κB organizes energy metabolism networks by controlling the balance between the utilization of glycolysis and mitochondrial respiration. NF-κB inhibition causes cellular reprogramming to aerobic glycolysis under basal conditions and induces necrosis on glucose starvation. The metabolic reorganization that results from NF-κB inhibition overcomes the requirement for tumour suppressor mutation in oncogenic transformation and impairs metabolic adaptation in cancer in vivo. This NF-κB-dependent metabolic pathway involves stimulation of oxidative phosphorylation through upregulation of mitochondrial synthesis of cytochrome c oxidase 2 (SCO2; ref. ). Our findings identify NF-κB as a physiological regulator of mitochondrial respiration and establish a role for NF-κB in metabolic adaptation in normal cells and cancer.

title: p53-dependent regulation of mitochondrial energy production by the RelA subunit of NF-κB.
abstract: Aberrant activity of the nuclear factor kappaB (NF-κB) transcription factor family, which regulates cellular responses to stress and infection, is associated with many human cancers. In this study, we define a function of NF-κB in regulation of cellular respiration that is dependent upon the tumor suppressor p53. Translocation of the NF-κB family member RelA to mitochondria was inhibited by p53 by blocking an essential interaction with the HSP Mortalin. However, in the absence of p53, RelA was transported into the mitochondria and recruited to the mitochondrial genome where it repressed mitochondrial gene expression, oxygen consumption, and cellular ATP levels. We found mitochondrial RelA function to be dependent on its conserved C-terminal transactivation domain and independent of its sequence-specific DNA-binding ability, suggesting that its function in this setting was mediated by direct interaction with mitochondrial transcription factors. Taken together, our findings uncover a new mechanism through which RelA can regulate mitochondrial function, with important implications for how NF-κB activity and loss of p53 can contribute to changes in tumor cell metabolism and energy production.

title: Differential utilization of two ATP-generating pathways is regulated by p53.
abstract: A fundamental property of cancer cells is the preferential utilization of glycolysis over aerobic respiration to produce ATP. Renewed interest in understanding the mechanism underlying this metabolic shift in energy production is broadening our understanding of the relationship between cancer and cellular metabolism. In a recent article, Matoba et al. report that the p53 tumor suppressor regulates the expression of SCO2, a protein that is required for the assembly of cytochrome c oxidase (COX), a multimeric protein complex required for oxidative phosphorylation. The implication of these findings is that aerobic respiration is compromised in cells that lack functional p53.

title: p53 Improves Aerobic Exercise Capacity and Augments Skeletal Muscle Mitochondrial DNA Content
abstract: Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health.  The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity.  Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A (TFAM), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53+/+ compared to p53−/− mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content.  Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.

title: Mitochondrial dysfunction in Parkinson's disease
abstract: Parkinson's disease (PD) is the second most common neurodegenerative disease. About 2% of the population above the age of 60 is affected by the disease. The pathological hallmarks of the disease include the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies that are made of α‐synuclein. Several theories have been suggested for the pathogenesis of PD, of which mitochondrial dysfunction plays a pivotal role in both sporadic and familial forms of the disease. Dysfunction of the mitochondria that is caused by bioenergetic defects, mutations in mitochondrial DNA, nuclear DNA gene mutations linked to mitochondria, and changes in dynamics of the mitochondria such fusion or fission, changes in size and morphology, alterations in trafficking or transport, altered movement of mitochondria, impairment of transcription, and the presence of mutated proteins associated with mitochondria are implicated in PD. In this review, we provide a detailed overview of the mechanisms that can cause mitochondrial dysfunction in PD. We bring to the forefront, new signaling pathways such as the retromer‐trafficking pathway and its implication in the disease and also provide a brief overview of therapeutic strategies to improve mitochondrial defects in PD.

title: Cytopathic hypoxia in sepsis
abstract: Diminished availability of oxygen at the cellular level might account for organ dysfunction in sepsis. Although the classical forms of tissue hypoxia due to hypoxemia, anemia, or inadequate perfusion all might be important under some conditions, it seems increasingly likely that a fourth mechanism, namely cytopathic hypoxia, might play a role as well. The term cytopathic hypoxia is used to denote diminished production of adenosine triphosphate (ATP) despite normal (or even supranormal) PO2 values in the vicinity of mitochondria within cells. At least in theory, cytopathic hypoxia could be a consequence of several different (but mutually compatible) pathogenic mechanisms, including diminished delivery of a key substrate (e.g., pyruvate) into the mitochondrial tricarboxylic acid (TCA) cycle, inhibition of key mitochondrial enzymes involved in either the TCA cycle or the electron transport chain, activation of the enzyme, poly‐(ADP)‐ribosylpolymerase (PARP), or collapse of the protonic gradient across the inner mitochondrial membrane leading to uncoupling of oxidation (of NADH and FADH) from phosphorylation of ADP to form ATP. Tantalizing, but limited, data support the view that cytopathic hypoxia occurs in both animals and patients with sepsis or endotoxemia.

title: A global approach to energy metabolism in an experimental model of sepsis.
abstract: Disturbances in energy metabolism during sepsis are not clearly understood. The aim of the study was to globally assess the energy drive in septic rat myocytes, studying both glycolysis rates and mitochondrial maximal activities together, using recent in vitro techniques. Measurements were assessed before (H0) and 4 h after sepsis induction (H4). Hyperlactatemia was observed in all septic animals ([lactate] = 1.2 +/- 0.3 mmol/L at H0 versus 3.3 +/- 0.6 mmol/L at H4; p < 0.001). An enhanced glycolysis rate was observed in both aerobic ( J(A) = 7.2 +/- 0.9 at H0 versus 18.2 +/- 4.1 nmol glucose/min/g at H4; p < 0.05) and anaerobic ( J(B) = 7.5 +/- 1.2 at H0 versus 15.4 +/- 3.4 micromol glucose/min/g at H4; p < 0.05) fluxes, associated with a selective significant pyruvate-malate-dependent oxygen consumption rate decrease (V O(2)-PM = 0.144 +/- 0.008 at H0 versus 0.113 +/- 0.007 micromol O(2)/h/mg at H4; p < 0.05). This oxygen consumption decrease can be interpreted either as a complex I and/or a complex I-ubiquinone relation alteration. Our results are consistent with the hypothesis that an altered mitochondrial function during sepsis is responsible, at least in part, for hyperlactatemia, which is thus a consequence of an increased glycolysis rate.

title: Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration. Involvement in response to hypoxia/reoxygenation.
abstract: Nitric oxide (NO); superoxide anion (O2.d-); the reaction product of NO with O2.d-, peroxynitrite (ONOO-); and ischemia/reperfusion have all been reported to inhibit respiration in isolated mitochondria. However, the specific species involved in the inhibition of respiration in intact tissues are poorly understood.



O2 consumption in isolated cardiac muscle from bovine calf hearts was quantified by use of a Clark-type electrode. Exogenous and endogenous sources of NO, from S-nitroso-N-acetylpenicillamine (SNAP) and bradykinin or carbachol, reversibly inhibited respiration, whereas the O2.- releasing agent, pyrogallol (PG), inhibited respiration in a manner that was only partially reversed when examined 15 minutes after the removal of PG. The generation of ONOO- with SNAP + PG caused a potentiation of the O2(-)-elicited inhibition of respiration when examined 15 minutes after the removal of the ONOO- generating system. Tiron (a scavenger of O2.-) did not alter the actions of SNAP, but it attenuated the direct inhibitory effects of PG +/- SNAP and essentially eliminated the suppression of respiration observed 15 minutes after removal of the O2.- or ONOO- generating system. Urate (a scavenger of ONOO-) antagonized only the actions of PG + SNAP. After exposure of muscle slices to a model of hypoxia (15 minutes) and reoxygenation (10 minutes), respiratory inhibition was observed. This reoxygenation-induced inhibition was potentiated by L-arginine, the substrate for NO biosynthesis, and was markedly blocked by nitro-L-arginine (an NO synthase inhibitor), Tiron, or urate.



The potentially physiological reversible regulation of respiration in cardiac muscle by NO is converted to an effect that does not show rapid reversibility under conditions in which ONOO- forms, and this could contribute to cardiac dysfunction in situations such as hypoxia/reoxygenation.

title: Nitric oxide potently and reversibly deenergizes mitochondria at low oxygen tension.
abstract: Nitric oxide (nitrogen monoxide, NO) at low concentrations can potently deenergize isolated liver and brain mitochondria at oxygen concentrations that prevail in cells and tissues. Deenergization is observed when mitochondria utilize respiratory substrates such as pyruvate plus malate, succinate, or ascorbate plus tetramethylphenylenediamine, but not when mitochondria are energized with ATP. The extent and duration of deenergization is determined by the concentration of NO and oxygen, and the kind of respiratory substrate. The NO-induced changes of the mitochondrial energy state are transient and are paralleled by release and reuptake of mitochondrial calcium. These findings reveal a direct action of NO on the mitochondrial respiratory chain and suggest that NO exerts some of its physiological and pathological effects by deenergizing mitochondria.

title: TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction.
abstract: Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O(2)(*)(-)) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O(2)(*)(-) and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague-Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 micromol/kg bw, orally), and TNF+ Tempol (Temp; 300 micromol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O(2)(*)(-) production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.

title: Regulation of tumor necrosis factor-induced, mitochondria- and reactive oxygen species-dependent cell death by the electron flux through the electron transport chain complex I.
abstract: Tumor necrosis factor (TNF) induces a caspase-independent but mitochondria-dependent cell death process in the mouse fibrosarcoma cell line L929. Mitochondria actively participate in this TNF-induced necrotic cell death by the generation of mitochondrial reactive oxygen species (ROS). The aim of this study was to identify the mitochondrial components involved in TNF-induced production of ROS and their regulation by bioenergetic pathways. Therefore, we analyzed the bioenergetic characteristics in two metabolic L929 variants that exhibit different sensitivities to TNF. L929gln cells use glutamine as respiratory substrate and are far more susceptible to TNF-induced ROS generation and cell death as L929glc cells that use glucose as respiratory substrate. We show that the higher levels of reducing NAD(P)H equivalents, detected in the desensitized L929glc cells, do not cause diminished ROS generation. To the contrary, TNF increases the levels of NAD(P)H, probably altering complex I activity. A multiparameter analysis of electron flux through the mitochondrial electron transport chain, TNF-induced ROS levels, and cell death convincingly demonstrates a dependence of TNF signaling on complex I activity. Also, the sensitizing effect of glutamine metabolism correlates with an enhanced contribution of complex I to the overall electron flux. This participation of complex I activity in TNF-induced cell death is regulated by substrate availability rather than by a direct modification of complex I proteins. From the results presented in this paper we conclude that TNF-induced ROS generation and cell death are strongly regulated by bioenergetic pathways that define electron flux through complex I of the electron transport chain.

title: In vivo TNF-alpha inhibition ameliorates cardiac mitochondrial dysfunction, oxidative stress, and apoptosis in experimental heart failure.
abstract: Heart failure is associated with increased myocardial expression of TNF-alpha. However, the role of TNF-alpha in the development of heart failure is not fully understood. In the present study, we investigated the contribution of TNF-alpha to myocardial mitochondrial dysfunction, oxidative stress, and apoptosis in a unique dog model of heart failure characterized by an activation of all of these pathological processes. Male mongrel dogs were randomly assigned (n = 10 each) to 1) normal controls; 2) chronic pacing (250 beats/min for 4 wk) with concomitant administration of etanercept, a soluble p75 TNF receptor fusion protein, 0.5 mg/kg subcutaneously twice weekly; 3) chronic pacing with administration of saline vehicle. Mitochondrial function was assessed by left ventricular (LV) tissue mitochondrial respiratory enzyme activities. Oxidative stress was assessed with aldehyde levels, and apoptosis was quantified by photometric enzyme immunoassay for cytoplasmic histone-associated DNA fragments and terminal deoxynucleotide transferase-mediated nick-end labeling (TUNEL) assays. LV activity levels of mitochondrial respiratory chain enzyme complex III and V were reduced in the saline-treated dogs and restored either partially (complex III) or completely (complex V) in the etanercept-treated dogs. Aldehyde levels, DNA fragments, and TUNEL-positive cells were increased in the saline-treated dogs and normalized in etanercept-treated dogs. These changes were accompanied by an attenuation of LV dilatation and partial restoration of ejection fraction. Our data demonstrate that TNF-alpha contributes to progressive LV dysfunction in pacing-induced heart failure, mediated in part by a local impairment in mitochondrial function and increase in oxidative stress and myocyte apoptosis.

title: Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.
abstract: Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.

title: Tumor necrosis factor alpha increases aerobic glycolysis and reduces oxidative metabolism in prostate epithelial cells
abstract: Chronic inflammation promotes prostate cancer formation and progression. Furthermore, alterations in energy metabolism are a hallmark of prostate cancer cells. However, the actions of inflammatory factors on the energy metabolism of prostate epithelial cells have not been previously investigated. This is the first study to report on the effect of the inflammatory cytokine tumor necrosis factor alpha (TNFα) on the glycolytic and oxidative metabolism, and the mitochondrial function of widely used prostate epithelial cells.

title: Tumor necrosis factor alpha induces Warburg‐like metabolism and is reversed by anti‐inflammatory curcumin in breast epithelial cells
abstract: The reprogramming of cellular metabolism in cancer cells is a well‐documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF‐10a) and malignant (MCF‐7) breast epithelial cells with low‐level (10 ng/ml) tumor necrosis factor alpha (TNF‐α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF‐α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF‐α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti‐inflammatory agent curcumin in a dose‐dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.

title: Increased activity and sensitivity of mitochondrial respiratory enzymes to tumor necrosis factor alpha-mediated inhibition is associated with increased cytotoxicity in drug-resistant leukemic cell lines.
abstract: The drug-resistant leukemic cell lines, CEM/VLB100 and K/DAU600, are more sensitive to tumor necrosis factor alpha (TNFalpha)-mediated cytotoxicity compared with their parental cell lines, CCRF-CEM and K562 cl.6. Drug-resistant leukemic cell lines have more active mitochondrial function, which is associated with a greater susceptibility to TNFalpha-induced respiratory inhibition. TNFalpha blocked electron transfer at three sites, NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and cytochrome c oxidase (complex IV). Respiratory rate and electron transport chain enzyme activities were significantly inhibited in the drug-resistant, TNF-sensitive cell lines. Respiratory inhibition preceded cell death by at least 5 to 8 hours. The respiratory failure was not compensated for by appropriate up-regulation of the glycolytic pathway. Increasing mitochondrial respiratory rate and enzyme activities by long-term culture with 2 mmol/L adenosine 5'-diphosphate (ADP) and Pi sensitized both drug-sensitive and drug-resistant cells to TNFalpha-induced cytolysis. Intramitochondrial free radicals generated by paraquat only had a limited and delayed effect on respiratory inhibition and cytolysis in comparison with the effect of TNFalpha. We conclude that TNFalpha-induced cytotoxicity in leukemic cells is, at least in part, mediated by inhibition of mitochondrial respiration. Free radical generation by TNFalpha may not directly lead to the observed inhibition of the mitochondrial electron transport and other mechanisms must be involved.

title: Bcl-xL Prevents the Initial Decrease in Mitochondrial Membrane Potential and Subsequent Reactive Oxygen Species Production during Tumor Necrosis Factor Alpha-Induced Apoptosis
abstract: The Bcl-2 family of proteins are involved in regulating the redox state of cells. However, the mode of action of Bcl-2 proteins remains unclear. This work analyzed the effects of Bcl-xL on the cellular redox state after treatment with tumor necrosis factor alpha (TNF-α) or exogenous oxidants. We show that in cells that undergo TNF-α-induced apoptosis, TNF-α induces a partial decrease in mitochondrial membrane potential (ΔΨ m ) followed by high levels of reactive oxygen species (ROS). ROS scavengers delay the progression of mitochondrial depolarization and apoptotic cell death. This indicates that ROS are important mediators of mitochondrial depolarization. However, ROS scavengers fail to prevent the initial TNF-α-induced decrease in ΔΨ m . In contrast, expression of Bcl-xL prevents both the initial decrease in ΔΨ m following TNF-α treatment and the subsequent induction of ROS. Bcl-xL itself does not act as a ROS scavenger. In addition, Bcl-xL does not block the initial decrease in ΔΨ m following treatment with the oxidant hydrogen peroxide. However, unlike control-transfected cells, Bcl-xL-expressing cells can recover their mitochondrial membrane potential following the initial drop in ΔΨ m induced by hydrogen peroxide. These data suggest that Bcl-xL plays a regulatory role in controlling the membrane potential of and ROS production by mitochondria rather than acting as a direct antioxidant.

title: EAPF/Phafin-2, a novel endoplasmic reticulum-associated protein, facilitates TNF-α-triggered cellular apoptosis through endoplasmic reticulum-mitochondrial apoptotic pathway
abstract: We recently identified the Phafin protein family, whose members all contain an N-terminal PH domain (pleckstrin homology) and a C-terminal FYVE (Fab1, YGLO23, Vps27, and EEA1) domain. LAPF (lysosome-associated apoptosis-inducing protein containing PH and FYVE domains, also known as Phafin-1), as one representative member of this new family, has been shown to be able to initiate caspase-independent apoptosis through lysosomal-mitochondrial apoptotic pathway. Here, we describe the cloning and functional characterization of another Phafin member, EAPF (endoplasmic reticulum-associated apoptosis-involved protein containing PH and FYVE domains)/Phafin-2. Overexpression of EAPF/Phafin-2 enhances the sensitivity of L929 and MCF-7 cells to TNF-α-induced apoptosis, concomitant with its partial translocation to endoplasmic reticulum (ER). Both the PH and the FYVE domains contribute to the ER translocation of EAPF/Phafin-2 as well as EAPF/Phafin-2-enhanced apoptosis. Knockdown of mouse and human EAPF/Phafin-2 expression protects L929 cells and MCF-7 cells from TNF-α-induced apoptosis, respectively. We demonstrate that EAPF/Phafin-2 induces a much sharper and more rapid Ca2+ influx triggered by TNF-α and Ca2+ release ER contributes to the enhancement of EAPF/Phafin-2 in TNF-induced apoptosis. EAPF/Phafin-2 increases the activity of caspase 12, suggesting that EAPF/Phafin-2 is involved in ER-related apoptotic pathway. Overexpression of EAPF/Phafin-2 also enhances TNF-α-induced activity of caspase 3 (but not caspase 8 or 9), and promotes TNF-α-triggered mitochondrial membrane permeabilization (MMP) in L929 cells, including dissipation of mitochondrial membrane potential and release of AIF. Besides, EAPF/Phafin-2 also suppresses the unfolded protein response by inhibiting phosphorylation of eIF2α. Therefore, our results demonstrate that EAPF/Phafin-2 facilitates TNF-α-induced cellular apoptosis through an ER-mitochondrial apoptotic pathway, which may improve our understanding of drug-induced cancer cell death and cancer chemotherapy.

title: Involvement of the mitochondrion respiratory chain in the synergy achieved by treatment of human ovarian carcinoma cell lines with both tumor necrosis factor‐α and cis‐diamminedichloroplatinum
abstract: Previous studies have demonstrated that treatment of human tumor cell lines with a combination of cis‐diamminedichloroplatinum (CDDP) and tumor necrosis factor‐α (TNF‐α) results in additive/synergistic cytotoxic effects and reverses tumor cell resistance to TNF drugs. Free radical intermediates are induced by both TNF‐α and CDDP; however, the role of free radicals in synergy is not known. This study investigated the effect of two inhibitors on synergy, phenoxan (Phe) and butylated hydroxyanisole (BHA), which inhibit Complex I and Complex I and II of the mitochondrion respiratory chain, respectively.

title: Tumour necrosis factor-alpha induces superoxide anion generation in mitochondria of L929 cells.
abstract: Within a few minutes after addition to L929 cells, tumour necrosis factor-alpha (TNF alpha) induced an increase in lucigenin-enhanced chemiluminescence that could be inhibited by superoxide dismutase. The generation of superoxide anion (O2.-) was sensitive to treatment with rotenone, antimycin A and cyanide, indicating that the signal originated from mitochondria. The mechanism of production of O2.- was shown to be independent of ATP synthesis, as uncoupling of this event from mitochondrial electron transport did not alter the generation of O2.- induced by TNF alpha. Chemiluminescence was further dependent on the presence of extracellular calcium, suggesting a role for this cation as a second messenger. This hypothesis was supported by the finding that inhibition of mitochondrial calcium uptake by Ruthenium Red exerted a protective effect on TNF alpha-treated L929 cells. Increased O2.- generation was followed by a marked decrease in mitochondrial dehydrogenase activity and cellular ATP levels, while cell membrane permeability was moderately increased. A role for mitochondrial O2.- generation in TNF alpha cytotoxicity was further supported by the finding that resistant L929 cells had decreased ability to produce O2.- in response to TNF alpha. In addition, we detected a decreased activity of the mitochondrial enzyme succinate dehydrogenase in these cells, suggesting that this component of the respiratory chain might be an important contributor to the TNF alpha-induced generation of O2.-.

title: TNF-alpha and IL-1 alpha inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: evidence for primary impairment of mitochondrial function.
abstract: Cytokines such as tumor necrosis factor alpha (TNF alpha) and Interleukin-1alpha (IL1alpha) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF alpha and IL1alpha. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF alpha (1, 10, 100, 1000 I.U./ml) and IL1alpha (0.1, 1, 10, 100 I.U./ml). TNF alpha- and IL1alpha- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF alpha and IL1alpha and a subsequent down regulation of PDH activity.

title: Role of interleukin 1 and tumor necrosis factor on energy metabolism in rabbits.
abstract: A study of the combined effects of intravenous infusion of the recombinant cytokines beta-interleukin 1 (IL-1) and alpha-tumor necrosis factor (TNF) on energy substrate metabolism in awake, conditioned, adult rabbits was performed. After a 2-h basal or control period, 48-h fasted rabbits were administered TNF and IL-1 as a bolus (5 micrograms/kg) followed by a continuous intravenous infusion (25 ng.kg-1.min-1) for 3 h. Significant increases in plasma lactate (P less than 0.01), glucose (P less than 0.01), and triglycerides (P less than 0.05) occurred during the combined infusion of IL-1 and TNF, whereas neither cytokine alone had no effect. There was a 33% increase in the rate of glucose appearance (P less than 0.05), but glucose clearance was not altered compared with the control period. Glucose oxidation increased during the combined cytokine infusion period and glucose recycling increased by 600% (P less than 0.002). Lactic acidosis and decreased oxygen consumption, as a result of the cytokine infusions, indicated development of anaerobic glycolytic metabolism. A reduction in the activity state of hepatic mitochondrial pyruvate dehydrogenase (65 vs. 82% in control animals, P less than 0.05) was consistent with the observed increase in anaerobic glycolysis. Thus the combined infusion of IL-1 and TNF in rabbits produces metabolic manifestations seen in severe injury and sepsis in human patients and, as such, may account for the profound alterations of energy metabolism seen in these conditions.