An evaluation of the recognised systemic inflammatory biomarkers of chronic sub-optimal inflammation provides evidence for inflammageing (IFA) during multiple sclerosis (MS).

The pathogenesis of the human demyelinating disorder multiple sclerosis (MS) involves the loss of immune tolerance to self-neuroantigens. A deterioration in immune tolerance is linked to inherent immune ageing, or immunosenescence (ISC). Previous work by the author has confirmed the presence of ISC during MS. Moreover, evidence verified a prematurely aged immune system that may change the frequency and profile of MS through an altered decline in immune tolerance. Immune ageing is closely linked to a chronic systemic sub-optimal inflammation, termed inflammageing (IFA), which disrupts the efficiency of immune tolerance by varying the dynamics of ISC that includes accelerated changes to the immune system over time. Therefore, a shifting deterioration in immunological tolerance may evolve during MS through adversely-scheduled effects of IFA on ISC. However, there is, to date, no collective proof of ongoing IFA during MS. The Review addresses the constraint and provides a systematic critique of compelling evidence, through appraisal of IFA-related biomarker studies, to support the occurrence of a sub-optimal inflammation during MS. The findings justify further work to unequivocally demonstrate IFA in MS and provide additional insight into the complex pathology and developing epidemiology of the disease.

The influence and impact of ageing and immunosenescence (ISC) on adaptive immunity during multiple sclerosis (MS) and the animal counterpart experimental autoimmune encephalomyelitis (EAE).

The human ageing process encompasses mechanisms that effect a decline in homeostasis with increased susceptibility to disease and the development of chronic life-threatening illness. Increasing age affects the immune system which undergoes a progressive loss of efficiency, termed immunosenescence (ISC), to impact on quantitative and functional aspects of innate and adaptive immunity. The human demyelinating disease multiple sclerosis (MS) and the corresponding animal model experimental autoimmune encephalomyelitis (EAE) are strongly governed by immunological events that primarily involve the adaptive arm of the immune response. MS and EAE are frequently characterised by a chronic pathology and a protracted disease course which thereby creates the potential for exposure to the inherent, on-going effects and consequences of ISC. Collective evidence is presented to confirm the occurrence of established and unendorsed biological markers of ISC during the development of both diseases. Moreover, results are discussed from studies during the course of MS and EAE that reveal a premature upregulation of ISC-related biomarkers which indicates untimely alterations to the adaptive immune system. The effects of ISC and a prematurely aged immune system on autoimmune-associated neurodegenerative conditions such as MS and EAE are largely unknown but current evaluation of data justifies and encourages further investigation.

Defining and Regulating Acute Inflammatory Lesion Formation during the Pathogenesis of Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

The primary pathology of the human central nervous system disease multiple sclerosis (MS) and the animal counterpart experimental autoimmune encephalomyelitis (EAE) includes immunological and inflammatory events. Immune system involvement in MS has been widely debated but the role of inflammation has received less attention. Classic acute inflammation features vasculitis, resident tissue macrophage and mast cell participation plus the involvement of circulatory-derived neutrophils and platelets. Pre-lesion development in MS incorporates cerebral vasculitis, activated resident microglia in normal appearing white matter together with infiltrating cell types and factors indicative of an acute inflammatory reaction. Similarly, the formation of perivascular lesions during early EAE includes characteristic neurovasculitis, the participation of central nervous system microglial phenotypes plus haemopoietic cells and mediators, signifying an ongoing acute inflammatory response. EAE has been extensively used as a screen to select drugs for MS treatment but has been criticised as unrepresentative of the human condition due to fundamental differences in disease induction and pathogenesis. The review provides compelling evidence for a distinct acute inflammatory phase in MS lesion formation that is convincingly reproduced in early EAE pathology. Moreover, consideration of drug efficacy studies undertaken during initial EAE validates the occurrence of an acute inflammatory phase in disease pathogenesis. Critical appraisal, recognition and acceptance of the mutual acute inflammatory components inherent in the primary pathology of MS and EAE reveals new targets and encourages confident and reliable employment of the animal model in the assessment of novel compounds for the control of key primary pathological events in human demyelinating disease.

Serum levels of 25-hydroxy vitamin D in normal Biozzi and C57BL/6 mice and during the course of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE).

OBJECTIVE AND DESIGN: The aim of the study was to examine possible variations in the levels of 25-hydroxy vitamin D [25-(OH)D] in sera from normal Biozzi and C57BL/6 mice and during the course of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE). MATERIAL: Serum concentrations of 25-(OH)D were measured in normal male and female Biozzi and C57BL/6 mice, at 3-4 weeks old and 8-10 weeks old, with a minimum of six animals/group. Levels of the vitamin were also determined in CR EAE-inoculated mice, and controls, during the course of the disease using a minimum of six animals/treatment. METHODS: Cardiac blood was collected from the groups of normal, control and CR EAE-sensitised mice and sera prepared, by centrifugation of clotted samples, and assayed for 25-(OH)D levels by chemiluminescence assay. RESULTS: Normal male and female Biozzi and C57BL/6 mice had significantly higher levels of 25-(OH)D at 8-10 weeks old compared to concentrations at 3-4 weeks of age (P < 0.005). Also, levels of the vitamin were significantly raised in C57BL/6 male and female mice compared to values in samples from corresponding Biozzi mice. In addition, the amounts of 25-(OH)D in sera from female Biozzi and C57BL/6 mice were significantly increased compared to strain and aged-matched male mice. The CR EAE mice with acute stage disease had significantly higher 25-(OH)D levels compared to controls (P < 0.005). Vitamin concentrations fell to within controls values with the progression of CR EAE. CONCLUSIONS: Our preliminary studies have revealed marked differences between the amounts of 25-(OH)D in sera from Biozzi and C57BL/6 mice together with clear gender bias. The investigations also show significant, but selective changes, in levels of the vitamin during the course of CR EAE that are not always associated with the neurological disease state.

N-Methyl-D-aspartate (NMDA) receptor involvement in central nervous system prostaglandin production during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE).

Our previous studies have established that major changes in central nervous system (CNS) prostaglandin (PG) levels occur during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE), an animal model of the human demyelinating disease multiple sclerosis. PG production is controlled through a series of enzymic pathways that, in EAE, are influenced by neuroantigen-driven autoimmune events. In non-immune-based models of CNS disease, endogenous glucocorticoids have been proposed as instigators of PG synthesis via activation of the N-methyl-D-aspartate (NMDA) receptor. Glucocorticoids have an important regulatory role in the pathogenesis EAE and the NMDA receptor is intimately involved in many of the characteristic neuroinflammatory processes that govern the disease. Therefore, the alterations in prostanoid concentrations during the relapse stage of CR EAE may ultimately be governed by glucocorticoid-induced NMDA receptor activation. The current investigation has examined the proposed glucocorticoid-NMDA receptor link by determining the effects of the receptor antagonist, (+) MK-801, on CNS PGE 2 and PGD 2 levels in Biozzi mice with relapse symptoms of CR EAE. Prostanoid concentrations in the cerebral cortex were not altered by drug administration, and in cerebellar tissues, a vehicle effect negated any drug-induced changes. However, the level of PGD 2 in spinal cords from (+) MK-801-dosed mice was significantly lower, compared to controls, but PGE 2 concentrations remained unchanged. The results suggest that glucocorticoid-NMDA receptor-linked events are not primarily responsible for PG generation in the brain but may influence prostanoid production in discrete areas of the CNS.

Paracetamol-induced hypothermia is independent of cannabinoids and transient receptor potential vanilloid-1 and is not mediated by AM404.

In recent years, there has been increasing interest in hypothermia induced by paracetamol for therapeutic purposes, which, in some instances, has been reported as a side effect. Understanding the mechanism by which paracetamol induces hypothermia is therefore an important question. In this study, we investigated whether the novel metabolite of paracetamol, N-(4-hydroxyphenyl)arachidonylamide (AM404), which activates the cannabinoid (CB) and transient receptor potential vanilloid-1 (TRPV1) systems, mediates the paracetamol-induced hypothermia. The hypothermic response to 300 mg/kg paracetamol in CB(1) receptor (CB(1)R) and TRPV1 knockout mice was compared to wild-type mice. Hypothermia induced by paracetamol was also investigated in animals pretreated with the CB(1)R or TRPV1 antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperdinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251) or 4'-chloro-3-methoxycinnamanilide (SB366791), respectively. In CB(1)R or TRPV1 knockout mice, paracetamol induced hypothermia to the same extent as in wild-type mice. In addition, in C57BL/6 mice pretreated with AM251 or SB366791, paracetamol induced hypothermia to the same extent as in control mice. AM404 failed to induce hypothermia at pharmacological doses. Inhibition of fatty acid amide hydrolase (FAAH), which is involved in the metabolism of paracetamol to AM404, did not prevent the development of hypothermia with paracetamol. Paracetamol also induced hypothermia in FAAH knockout mice to the same extent as wild-type mice. We conclude that paracetamol induces hypothermia independent of cannabinoids and TRPV1 and that AM404 does not mediate this response. In addition, potential therapeutic value of combinational drug-induced hypothermia is supported by experimental evidence.

Cyclooxygenase expression and prostaglandin levels in central nervous system tissues during the course of chronic relapsing experimental autoimmune encephalomyelitis (EAE).

OBJECTIVE: Multiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and orchestrates both diseases. One particular group of mediators are the prostaglandins (PGs), which we have previously shown, through quantitation and pharmacological intervention, to be closely involved in the pathology of MS and EAE. The aim of the current study was to determine the expression of the PG-generating cyclooxygenase (COX) enzymes and the profile of PGE(2) and PGD(2), in selected central nervous system (CNS) tissues, with the development of the chronic relapsing (CR) form of EAE. In particular, the work investigates the possible relationship between the expression of COX isoenzymes and PG levels during the neurological phases of CR EAE. METHODS: CR EAE was induced in Biozzi mice with inoculum containing lyophilised, syngeneic spinal cord emulsified in complete Freund's adjuvant. The cerebral cortex, cerebellum and spinal cord were dissected from mice during the acute, remission and relapse stages of disease with a minimum of five animals per treatment. The expression of COX-1, COX-1b variant and COX-2, in pooled samples, was determined by Western blotting. PGE(2) and PGD(2) levels in extracted samples were measured using commercial enzyme immunoassay kits. RESULTS: COX-2 expression in spinal cords during acute disease remained unaltered and was in contrast to an enhancement of the enzyme, together with COX-1 and COX-1b, in all other sampled areas. PGE(2) and PGD(2) levels remained unchanged during the acute phase and the subsequent remission of symptoms. COX-1 and COX-1b expression was elevated in tissues during the relapse stage of CR EAE and concentrations of the prostanoids were markedly increased. CONCLUSIONS: The study examines the implications of COX isoenzyme expression over the course of CR EAE and discusses the reported relationship between PGE(2) and PGD(2) in the instigation and resolution of CNS inflammation. Consideration is also given to the treatment of CR EAE and suggests that drugs designed to limit the inflammatory effects of the PGs should be administered prior to or during the relapse phase of the disease.

A comparative evaluation of the response to peroxynitrite by a brain endothelial cell line and control of the effects by drug targeting.

The potent oxidant peroxynitrite (ONOO(-)) is formed after the combination of nitric oxide with superoxide and has been closely associated with the pathology of inflammatory disease. In particular, the generation of ONOO(-) has been linked to central nervous system disorders including Alzheimer's and Parkinson's disease, multiple sclerosis and bacterial and viral meningitis. Specifically, ONOO(-) has been implicated in the loss of blood-brain barrier (BBB) integrity during neuroinflammation, but the precise mechanisms through which the molecule acts to mediate neurovascular breakdown have not been established. The disruptive effects of ONOO(-) could be mediated by either direct or indirect actions on the endothelial cells that comprise the major component of the BBB. The current study has comparatively assessed the direct toxic effects of ONOO(-) on the brain endothelial cell line, b.End3 and C6 astrocytoma and NA neuroblastoma preparations. b.End3 cells were relatively resistant to ONOO(-)-induced cell death compared with C6 and NA cultures. The indirect involvement of ONOO(-) in neuroendothelial disruption was pharmacologically determined via adhesion molecule expression and immunocompetent cell attachment to b.End3 cells. ONOO(-)-targeted drugs, including the selective free radical scavenger, uric acid, the decomposition catalyst 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinatoiron (III) (FeTPPS) and the poly(ADP-ribose) polymerase inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino) acetamide hydrochloride (PJ34) revealed that ONOO(-) was only partly involved in E-selectin, ICAM-1 and VCAM-1 expression on b.End3 cells and also cytokine-induced T-lymphocyte attachment to the cell line. The results indicate that ONOO(-) contributes to b.End3 cell disruption but is not exclusively responsible for the breakdown of neuroendothelial function.

The acute and chronic phases of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE) are ameliorated by the peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron (III) chloride, (FeTPPS).

There is increasing evidence that the oxidative radical, peroxynitrite (ONOO(-)), is involved in the pathogenesis of inflammatory diseases including multiple sclerosis and the animal counterpart, experimental autoimmune encephalomyelitis (EAE). Compounds that impede the actions of ONOO(-) have proved useful in the control of EAE. In particular, catalytic isomerisation of ONOO(-) to inactive nitrate, through the use of metalloporphyrins, curtails the cellular response to inflammatory stimuli and halts the progression of neuroinflammation during EAE. The present study examined the pharmacological effects of the metalloporphyrin and ONOO(-) decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron(III)chloride (FeTPPS) on the acute and relapse phases of chronic relapsing (CR) EAE. Administration of FeTPPS to CR EAE-inoculated Biozzi mice commenced either therapeutically and immediately prior to the emergence of acute or relapse symptoms, or prophylactically, from the onset of remission of acute neurological signs. Drug therapy reduced acute and relapse symptoms but, and in contrast to the former phase, was of limited benefit in preventing histological changes during the latter stage of disease. In contrast, prophylactic FeTPPS was effective in limiting CNS pathology and neurological deficits. The findings confirm the inhibitory effects of FeTPPS on acute stage EAE. Moreover, the study extends previous observations by verifying compound efficacy on relapsing disease. Use of metalloporphyrins, such as FeTPPS, again highlights the important role played by ONOO(-) in the development of inflammatory diseases such as EAE.

Glutamate receptors in neuroinflammatory demyelinating disease.

Multiple sclerosis (MS) is a chronic demyelinating disease of the human central nervous system (CNS). The condition predominantly affects young adults and is characterised by immunological and inflammatory changes in the periphery and CNS that contribute to neurovascular disruption, haemopoietic cell invasion of target tissues, and demyelination of nerve fibres which culminate in neurological deficits that relapse and remit or are progressive. The main features of MS can be reproduced in the inducible animal counterpart, experimental autoimmune encephalomyelitis (EAE). The search for new MS treatments invariably employs EAE to determine drug activity and provide a rationale for exploring clinical efficacy. The preclinical development of compounds for MS has generally followed a conventional, immunotherapeutic route. However, over the past decade, a group of compounds that suppress EAE but have no apparent immunomodulatory activity have emerged. These drugs interact with the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-isoxazolepropionic acid (AMPA)/kainate family of glutamate receptors reported to control neurovascular permeability, inflammatory mediator synthesis, and resident glial cell functions including CNS myelination. The review considers the importance of the glutamate receptors in EAE and MS pathogenesis. The use of receptor antagonists to control EAE is also discussed together with the possibility of therapeutic application in demyelinating disease.

Efficacy, safety, and pharmacokinetics of levobupivacaine with and without fentanyl after continuous epidural infusion in children: a multicenter trial.

BACKGROUND: Levobupivacaine, the levo-enantiomer of bupivacaine, is as potent as bupivacaine but less toxic. Therefore, the authors investigated the efficacy, safety, and pharmacokinetics of perioperative epidural levobupivacaine with and without fentanyl in children. METHODS: After Research Ethics Board approval and informed written consent, 120 healthy children aged 6 months to 12 yr who were scheduled to undergo urologic or abdominal surgery were randomized in a double-blinded and concealed manner to receive one of four epidural solutions as a continuous infusion for 24 h: 0.125% levobupivacaine; 0.0625% levobupivacaine; 1 mug/ml fentanyl; or the combination, 0.0625 levobupivacaine and 1 mug/ml fentanyl. After induction of anesthesia and tracheal intubation, a lumbar epidural catheter was sited, a loading dose was administered (0.75 ml/kg levobupivacaine, 0.175%), and the epidural infusion was commenced. The primary endpoint was the need for rescue analgesia (morphine) in the first 10 h after surgery. Pain, motor strength, and side effects were recorded for 24 h. Venous blood was collected from 18 children to determine the plasma concentrations of levobupivacaine and/or fentanyl before and 2, 4, 8, 16, 24, and 26 or 30 h after the start of the epidural infusion. RESULTS: Of the 114 children who were analyzed for intention to treat, a similar number of children in each group reached the 10-h mark. The time to the first dose of morphine in the first 10 h was less in the plain fentanyl group (P < 0.044). All other effects were similar among the four groups. The plasma concentration of levobupivacaine increased during the infusion period, reaching a maximum of 0.76 +/- 0.11 mug/ml in the 0.125% group and 0.48 +/- 0.12 mug/ml in the 0.0625% group by 24 h. The plasma concentration of fentanyl also increased steadily, reaching a maximum concentration of 0.37 +/- 0.11 ng/ml by 24 h. CONCLUSION: We conclude that 0.0625% levobupivacaine without fentanyl is an effective perioperative epidural solution in children when infused at a rate of 0.3 ml. kg-1. h-1. The plasma concentrations of 0.125% and 0.0625% levobupivacaine and fentanyl (1 mug/ml) at the end of a 24-h infusion are low.

Modulation of blood-brain barrier dysfunction and neurological deficits during acute experimental allergic encephalomyelitis by the N-methyl-D-aspartate receptor antagonist memantine.

Previous studies by us have strongly indicated a role for the N-methyl-D-aspartate (NMDA) receptor in the pathogenesis of experimental allergic encephalomyelitis (EAE) and, moreover, the loss of blood-brain barrier (BBB) integrity implicit in the disease. The current investigation has used the NMDA receptor antagonist memantine to modify the neurological course of EAE and, in particular, prevent BBB breakdown. Memantine was administered orally either semiprophylactically, from day 7 postinoculation (PI), or therapeutically, 10 to 11 days PI. Semiprophylactic administration of drug at 60 mg/kg b.wt. significantly restored BBB integrity, reduced symptoms, and limited inflammatory lesions (p < 0.05), when assessed 12 days PI. Higher concentrations of memantine did not notably advance disease improvements observed at 60 mg/kg b.wt., and 40-mg/kg b.wt. doses only reduced histological scores (p < 0.05). Therapeutic application of memantine was found to be as effective as semiprophylactic dosing. Administration of drug at 60 mg/kg b.wt. was demonstrated as the optimum dose, significantly reducing disease, BBB permeability, and lesions (p < 0.01). Extended studies revealed that, after cessation of memantine treatment using either dosing regime, any subsequent appearance of disease was suppressed in severity and duration. We have provided further strong evidence in support of a role for the NMDA receptor in the development of EAE and, in particular, the loss of BBB function and recruitment of inflammatory cells. Moreover, memantine is therapeutically efficacious, suggesting the NMDA receptor as a viable pharmacological target for future treatment of human neurological conditions such as multiple sclerosis.