The contribution of the acute phase response to the pathogenesis of relapse in chronic-relapsing experimental autoimmune encephalitis models of multiple sclerosis.

BACKGROUND: Increased relapse rates in multiple sclerosis (MS) as a consequence of peripheral immune system activation, owing to infection for example, have been widely reported, but the mechanism remains unclear. Acute brain injury models can be exacerbated by augmenting the hepatic acute phase response (APR). Here, we explored the contribution of the hepatic APR to relapse in two rodent models of MS. METHODS: Mice with MOG-CFA-induced chronic relapsing experimental autoimmune encephalitis (CR-EAE) were killed before, during and after the first phase of disease, and the brain and liver chemokine, cytokine and acute phase protein (APP) mRNA expression profile was determined. During remission, the APR was reactivated with an intraperitoneal lipopolysaccharide (LPS) and clinical score was monitored throughout. To explore the downstream mediators, CXCL-1, which is induced as part of the APR, was injected into animals with a focal, cytokine/MOG-induced EAE lesion (fEAE) and the cellularity of the lesions was assessed. RESULTS: Compared to CFA control, in a rodent CR-EAE model, an hepatic APR preceded clinical signs and central cytokine production in the initial phase of disease. Compared to administration in naïve animals, an LPS challenge during the asymptomatic remission phase of CR-EAE rodents provoked relapse and resulted in the increased and extended expression of specific peripheral hepatic chemokines. CXCL-1 and several other APPs were markedly elevated. A single intravenous administration of the highly induced chemokine, CXCL-1, was found to be sufficient to reactivate the lesions by increasing microglial activation and the recruitment of T cells in fEAE lesions. CONCLUSIONS: The APR plays a contributing role to the pathology seen in models of chronic brain injury and in translating the effects of peripheral immune system stimulation secondary to trauma or infection into central pathology and behavioural signs. Further elucidation of the exact mechanisms in this process will inform development of more effective, selective therapies in MS that, by suppressing the hepatic chemokine response, may prevent relapse.

Viral pre-challenge increases central nervous system inflammation after intracranial interleukin-1ß injection.

INTRODUCTION: Systemic inflammation has been shown to significantly worsen the outcome of neurological disease. However, after acute injuries to the brain both pre- and post-conditioning with bacterial endotoxin has been shown to reduce leukocyte recruitment to the CNS. Here, we sought to determine whether viral pre-challenge would have an effect on the outcome of acute CNS inflammation that was distinct from endotoxin. METHODS: Animals received a single intracranial microinjection of IL-1ß in the presence or absence of a viral pre-challenge 24 hours prior to surgery. Liver and brain tissue were analysed for chemokine expression by qRT-PCR and leukocyte and monocyte infiltration 12 hours, 3 days and 7 days after the IL-1ß injection. RESULTS: Here, a single injection of adenovirus prior to IL-1ß injection resulted in adhesion molecule expression, chemokine expression and the recruitment of neutrophils to the injured CNS in significantly higher numbers than in IL-1ß injected animals. The distribution and persistence of leukocytes within the CNS was also greater after pre-challenge, with neutrophils being found in both the ipsilateral and contralateral hemispheres. Thus, despite the absence of virus within the CNS, the presence of virus within the periphery was sufficient to exacerbate CNS disease. CONCLUSIONS: These data suggest that the effect of a peripheral inflammatory challenge on the outcome of CNS injury or disease is not generic and will be highly dependent on the nature of the pathogen.

Anti-IL-17A treatment reduces clinical score and VCAM-1 expression detected by in vivo magnetic resonance imaging in chronic relapsing EAE ABH mice.

IL-17 is argued to play an important role in the multiple sclerosis-like disease experimental autoimmune encephalitis (EAE). We investigated the therapeutic effects of anti-IL-17A in a chronic relapsing EAE ABH mouse model using conventional scoring, quantitative behavioral outcomes, and a novel vascular cell adhesion molecule 1 (VCAM-1)-targeted magnetic resonance imaging (MRI) contrast agent [anti-VCAM-microparticles of iron oxide (MPIO)] to identify conventionally undetectable neuropathology. Mice were administered prophylactic or treatment regimens of anti-IL-17A or IgG and two injections of anti-VCAM-MPIO before undergoing T2*-weighted three-dimensional and gadolinium-diethylenetriamine pentaacetic acid T1-weighted MRI. Rotarod, inverted screen, and open field motor function tests were performed, conventional clinical scores calculated, and central IL-17A mRNA expression quantified during acute disease, remission, and relapse. Prophylactic anti-IL-17A prevents acute disease and relapse and is associated with reduced clinical and functional severity. Treatment regimens delay relapse, improve functional scores, and are associated with reduced VCAM-MPIO lesions during remission. No significant alteration was detectable in levels of gadolinium-diethylenetriamine pentaacetic acid- or VCAM-MPIO-positive lesions during relapse. Prophylactic and treatment anti-IL-17A were therapeutically effective in chronic relapsing EAE, improving clinical and quantifiable functional outcomes. IL-17A expression seems significant during acute disease but less important chronically. Disease-related immunoneuropathology is more sensitively detected using VCAM-MPIO MRI, which may, therefore, be used to monitor therapy meaningfully.

Expanding the diversity of chemical protein modification allows post-translational mimicry.

One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. We therefore anticipate that this ability to build model systems will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein's PTMs from an in vivo assay into an in vitro system.

VCAM-1-targeted magnetic resonance imaging reveals subclinical disease in a mouse model of multiple sclerosis.

Diagnosis of multiple sclerosis (MS) currently requires lesion identification by gadolinium (Gd)-enhanced or T(2)-weighted magnetic resonance imaging (MRI). However, these methods only identify late-stage pathology associated with blood-brain barrier breakdown. There is a growing belief that more widespread, but currently undetectable, pathology is present in the MS brain. We have previously demonstrated that an anti-VCAM-1 antibody conjugated to microparticles of iron oxide (VCAM-MPIO) enables in vivo detection of VCAM-1 by MRI. Here, in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we have shown that presymptomatic lesions can be quantified using VCAM-MPIO when they are undetectable by Gd-enhancing MRI. Moreover, in symptomatic animals VCAM-MPIO binding was present in all regions showing Gd-DTPA enhancement and also in areas of no Gd-DTPA enhancement, which were confirmed histologically to be regions of leukocyte infiltration. VCAM-MPIO binding correlated significantly with increasing disability. Negligible MPIO-induced contrast was found in either EAE animals injected with an equivalent nontargeted contrast agent (IgG-MPIO) or in control animals injected with the VCAM-MPIO. These findings describe a highly sensitive molecular imaging tool that may enable detection of currently invisible pathology in MS, thus accelerating diagnosis, guiding treatment, and enabling quantitative disease assessment.

Glyconanoparticles allow pre-symptomatic in vivo imaging of brain disease.

Initial recruitment of leukocytes in inflammation associated with diseases such as multiple sclerosis (MS), ischemic stroke, and HIV-related dementia, takes place across intact, but activated brain endothelium. It is therefore undetectable to symptom-based diagnoses and cannot be observed by conventional imaging techniques, which rely on increased permeability of the blood-brain barrier (BBB) in later stages of disease. Specific visualization of the early-activated cerebral endothelium would provide a powerful tool for the presymptomatic diagnosis of brain disease and evaluation of new therapies. Here, we present the design, construction and in vivo application of carbohydrate-functionalized nanoparticles that allow direct detection of endothelial markers E-/P-selectin (CD62E/CD62P) in acute inflammation. These first examples of MRI-visible glyconanoparticles display multiple copies of the natural complex glycan ligand of selectins. Their resulting sensitivity and binding selectivity has allowed acute detection of disease in mammals with beneficial implications for treatment of an expanding patient population suffering from neurological disease.

Selective blood-brain barrier permeabilization of brain metastases by a type 1 receptor-selective tumor necrosis factor mutein.

BACKGROUND: Metastasis to the brain is a major challenge with poor prognosis. The blood-brain barrier (BBB) is a significant impediment to effective treatment, being intact during the early stages of tumor development and heterogeneously permeable at later stages. Intravenous injection of tumor necrosis factor (TNF) selectively induces BBB permeabilization at sites of brain micrometastasis, in a TNF type 1 receptor (TNFR1)-dependent manner. Here, to enable clinical translation, we have developed a TNFR1-selective agonist variant of human TNF that induces BBB permeabilization, while minimizing potential toxicity. METHODS: A library of human TNF muteins (mutTNF) was generated and assessed for binding specificity to mouse and human TNFR1/2, endothelial permeabilizing activity in vitro, potential immunogenicity, and circulatory half-life. The permeabilizing ability of the most promising variant was assessed in vivo in a model of brain metastasis. RESULTS: The primary mutTNF variant showed similar affinity for human TNFR1 than wild-type human TNF, similar affinity for mouse TNFR1 as wild-type mouse TNF, undetectable binding to human/mouse TNFR2, low potential immunogenicity, and permeabilization of an endothelial monolayer. Circulatory half-life was similar to mouse/human TNF and BBB permeabilization was induced selectively at sites of micrometastases in vivo, with a time window of ≥24 hours and enabling delivery of agents within a therapeutically relevant range (0.5-150 kDa), including the clinically approved therapy, trastuzumab. CONCLUSIONS: We have developed a clinically translatable mutTNF that selectively opens the BBB at micrometastatic sites, while leaving the rest of the cerebrovasculature intact. This approach will open a window for brain metastasis treatment that currently does not exist.

A contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional MRI.

Human and murine cerebral malaria are associated with elevated levels of cytokines in the brain and adherence of platelets to the microvasculature. Here we demonstrated that the accumulation of platelets in the brain microvasculature can be detected with MRI, using what we believe to be a novel contrast agent, at a time when the pathology is undetectable by conventional MRI. Ligand-induced binding sites (LIBS) on activated platelet glycoprotein IIb/IIIa receptors were detected in the brains of malaria-infected mice 6 days after inoculation with Plasmodium berghei using microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody specific for the LIBS (LIBS-MPIO). No binding of the LIBS-MPIO contrast agent was detected in uninfected animals. A combination of LIBS-MPIO MRI, confocal microscopy, and transmission electron microscopy revealed that the proinflammatory cytokine TNF-alpha, but not IL-1beta or lymphotoxin-alpha (LT-alpha), induced adherence of platelets to cerebrovascular endothelium. Peak platelet adhesion was found 12 h after TNF-alpha injection and was readily detected with LIBS-MPIO contrast-enhanced MRI. Temporal studies revealed that the level of MPIO-induced contrast was proportional to the number of platelets bound. Thus, the LIBS-MPIO contrast agent enabled noninvasive detection of otherwise undetectable cerebral pathology by in vivo MRI before the appearance of clinical disease, highlighting the potential of targeted contrast agents for diagnostic, mechanistic, and therapeutic studies.

Systemic inflammatory response reactivates immune-mediated lesions in rat brain.

The potential association between microbial infection and reactivation of a multiple sclerosis (MS) lesion is an important issue that remains unresolved, primarily because of the absence of suitable animal models and imaging techniques. Here, we have evaluated this question in an empirical manner using immunohistochemistry and magnetic resonance imaging (MRI), before and after the induction of a systemic inflammatory response in two distinct models of MS. In a pattern-II-type focal myelin oligodendrocyte glycoprotein-experimental autoimmune encephalomyelitis model, systemic endotoxin injection caused an increase in regional cerebral blood volume (rCBV) around the lesion site after 6 h, together with a reduction in the magnetization transfer ratio of the lesioned corpus callosum. These changes were followed by an increase in the diffusion of tissue water within the lesion 24 h after endotoxin challenge and new leukocyte recruitment as revealed both immunohistochemically and by MRI tracking of ultrasmall superparamagnetic iron oxide-labeled macrophages. Importantly, we detected in vivo expression of E- and P-selectin in quiescent lesions by MRI-detectable glyconanoparticles conjugated to sialyl Lewis(X). This finding may explain, at least in part, the ability of quiescent MS lesions to rapidly reinitiate the cell recruitment processes. In a pattern-I-type delayed-type hypersensitivity response model, a similar effect of endotoxin challenge on rCBV was observed, together with delayed breakdown of the blood-brain barrier, showing that systemic infection can alter the pathogenesis of MS-like lesions regardless of lesion etiology. These findings will have important implications for the management and monitoring of individuals with MS.

Sickness behaviour is induced by a peripheral CXC-chemokine also expressed in multiple sclerosis and EAE.

Non-CNS chemokine production may contribute to previously unrecognised components of Multiple Sclerosis (MS) pathology. Here we show that IL-8, a neutrophil chemoattractant, is significantly increased in serum from individuals with MS, and that the rodent homolog of IL-8 (CXCL1) is expressed in the liver in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. The hepatic expression of CXCL1 in EAE is accompanied by neutrophil recruitment to the liver, and we show that this recruitment is a feature of post mortem liver tissue from MS patients, which is a previously unrecognised phenomenon. We speculated that the presence of peripheral CXC-chemokine expression might contribute to the sickness behaviours associated with MS, which are a significant contributor to morbidity. Peripheral, but not central, administration of CXCL1 to Wistar rats inhibited spontaneous activity in the open field and burrowing behaviour in a dose-dependent manner (5-45 microg). The expression of CXCL1 by the liver and the recruitment of neutrophils can be modelled by the intracerebral injection of IL-1beta. Here, we found that interferon-beta (IFN-beta) pretreatment significantly inhibited hepatic CXCL1 production and neutrophil recruitment to the liver induced by the microinjection of IL-1beta into the brain. Thus while the mechanism by which IFN-beta therapy suppresses disease in MS remains unclear, the data presented here suggests that the inhibition of hepatic chemokine synthesis may be a contributing factor.

Comparison of MRI signatures in pattern I and II multiple sclerosis models.

The majority of individuals with multiple sclerosis (MS) exhibit T-cell- and macrophage-dominated lesions (patterns I and II; as opposed to III and IV). These lesions, in turn, may be distinguished on the basis of whether or not there are immunoglobulin and complement depositions at the sites of active myelin destruction; such depositions are found exclusively in pattern II lesions. The main aim of this study was to determine whether pattern I and pattern II MS lesions exhibit distinct MRI signatures. We have used a recently described focal MOG-induced EAE model of the rat brain, which recapitulates many of the hallmarks of pattern II MS; we compared this with our previous work in a delayed type hypersensitivity model of a pattern I type lesion in the rat brain. Demyelinating lesions with extensive inflammation were generated, in which the T2-weighted signal was increased. Magnetisation transfer ratio (MTR) maps revealed loss and subsequent incomplete recovery of the structure of the corpus callosum, together with changes in tissue water diffusion and an associated increase in ventricle size. Notably, the MTR changes preceeded histological demyelination and may report on the processes leading to demyelination, rather than demyelination per se. Immunohistochemically, these MRI-detectable signal changes correlated with both inflammatory cell infiltration and later loss of myelin. Breakdown of the blood-brain barrier and an increase in the regional cerebral blood volume were also evident in and around the lesion site at the early stage of the disease. Interestingly, however, the MRI signal changes in this pattern II type MS lesion were remarkably consistent with those previously observed in a pattern I lesion. These findings suggest that the observed signal changes reflect the convergent histopathology of the two models rather than the underlying mechanisms of the disease.

Hepatic nuclear factor kappa B regulates neutrophil recruitment to the injured brain.

Acute brain injury is associated with induction of hepatic chemokine expression, which is an essential element in the subsequent recruitment of leukocytes to the damaged brain. To further understand the significance of the hepatic inflammatory response, we focused on nuclear factor (NF)-kappa B, a pivotal regulator of inflammation. Nondestructive real-time whole-body imaging was undertaken in the 3XNF-kappa B-luciferase mouse to monitor NF-kappa B activation. Acute brain injury induced by intracerebral injection of interleukin-1 provoked rapid activation of hepatic and CNS NF-kappa B, with only minimal changes in other organs. Elevated NF-kappa B in the brain was limited to the site of the lesion, whereas hepatic NF-kappa B was widespread. The function of NF-kappa B in this model was determined by monitoring leukocyte recruitment to the liver and brain of nf kappa b1 mice, which lack the anti-inflammatory p50:p50 NF-kappa B homodimer. Brain injury in the nf kappa b1 mice was associated with increased neutrophil recruitment to the liver and brain compared with wild-type mice, thereby confirming a regulatory role for the NF-kappa B system. To determine the role of hepatic NF-kappa B, it was selectively inhibited by intravenous adenoviral-mediated delivery of an I kappa B alpha super-repressor. This treatment significantly reduced the numbers of neutrophils recruited to the brain. In conclusion, acute brain injury is associated with rapid and robust activation of hepatic NF-kappa B, which is required for efficient mobilization of circulating leukocytes to the brain.

Liver Kupffer cells control the magnitude of the inflammatory response in the injured brain and spinal cord.

The CNS inflammatory response is regulated by hepatic chemokine synthesis, which promotes leukocytosis and facilitates leukocyte recruitment to the site of injury. To understand the role of the individual cell populations in the liver during the hepatic response to acute brain injury, we selectively depleted Kupffer cells (KC), using clodronate-filled liposomes, and assessed the inflammatory response following a microinjection of IL-1beta into the rat brain or after a compression injury in the spinal cord. We show by immunohistochemistry that KC depletion reduces neutrophil infiltration into the IL-1beta-injected brain by 70% and by 50% into the contusion-injured spinal cord. qRT-PCR analysis of hepatic chemokine mRNA expression showed that chemokine expression in the liver after brain injury is not restricted to a single cell population. In non-depleted rats, CXCL-10, IL-1beta, CCL-2, and MIP-1alpha mRNAs were increased up to sixfold more than in KC depleted rats. However, CXCL-1 and MIP-1beta were not significantly affected by KC depletion. The reduction in chemokine mRNA expression by the liver was not associated with decreased neutrophil mobilisation as might have been expected. These findings suggest that in response to CNS injury, KC mediated mechanisms are responsible for increasing neutrophil entry to the site of CNS injury, but that neutrophil mobilisation is dependent on other non-KC mediated events. However, the suppression of KC activity may prevent secondary damage after acute brain injury.

Immunomodulatory effects of etanercept in a model of brain injury act through attenuation of the acute-phase response.

TNF-alpha has proved to be a successful target in the treatment of many peripheral inflammatory diseases, but the same interventions worsen immune-mediated CNS disease. However, anti-TNF-alpha strategies may offer promise as therapy for non-immune CNS injury. In this study, we have microinjected IL-1beta or lipopolysaccharide (LPS) into the rat brain as a simple model of brain injury and have systemically administered the TNF-alpha antagonist etanercept to discover whether hepatic TNF-alpha, produced as part of the acute-phase response to CNS injury, modulates the inflammatory response in the brain. We report a significant reduction in neutrophil numbers recruited to the IL-1beta- or LPS-challenged brain as a result of TNF-alpha inhibition. We also show an attenuation in the levels of hepatic mRNA including TNF-alpha mRNA and of TNF-alpha-induced genes, such as the chemokines CCL-2, CXCL-5, and CXCL-10, although other chemokines elevated by the injury were not significantly changed. The reduction in hepatic chemokine synthesis results in reduced numbers of circulating neutrophils, and also a reduction in the numbers recruited to the liver as a consequence of brain injury. These findings suggest that TNF-alpha inhibitors may reduce CNS inflammatory responses by targeting the hepatic acute-phase response, and thus therapies for brain injury need not cross the blood-brain barrier to be effective.

Hdm2 recruits a hypoxia-sensitive corepressor to negatively regulate p53-dependent transcription.

The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Here we show that Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which we show here results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress.

Covalent assembly of nanoparticles as a peptidase-degradable platform for molecular MRI.

Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.

CINC-1 is an acute-phase protein induced by focal brain injury causing leukocyte mobilization and liver injury.

Following injury or infection, the liver releases acute-phase proteins (APP). After a severe focal injury, this systemic response can be excessive and may lead to multiorgan dysfunction (MODS). CINC-1 is a neutrophil chemoattractant, and we have now established that it also functions as an early APP after injury to the brain or to peripheral tissues. After induction of a focal inflammatory lesion in the brain, there is rapid hepatic and serum CINC-1 induction, which is associated with increases in neutrophil numbers within the liver and within the circulation. CINC-1-mediated recruitment of neutrophils to organs distant from the primary injury site may contribute to MODS. Indeed, we found that enzyme markers of liver tissue injury are increased in the serum following generation of a focal inflammatory lesion in the brain. Neutralization of CINC-1 in the periphery reversed brain-injury-induced neutrophil mobilization and inhibited recruitment of neutrophils to the brain and to the liver. Thus, a significant component of the hepatic acute-phase response is the release of chemokines by the liver, which act to amplify the inflammatory response and modulate the subsequent leukocytosis and secondary tissue damage. Hepatic CINC-1 synthesis following injury presents a novel focus for treatment of inflammation.

The effect of B-cell depletion in the Theiler's model of multiple sclerosis.

B cell depletion (BCD) is being considered as a treatment for multiple sclerosis (MS), but there are many uncertainties surrounding the use of this therapy, such as its potential effect in individuals with concurrent viral infections. We sought to discover what effect BCD, induced by an anti-CD20 monoclonal antibody, would have on Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Mice were injected with the anti-CD20 monoclonal antibody 5D2, 14 days before or 14 days after infection with TMEV. Efficacy of depletion of B cells was assessed by flow cytometry of CD19(+) cells. Mouse disability was measured by Rotarod, viral load was measured by real time PCR for TMEV RNA. Binding and neutralizing antibody levels were determined in sera and CSF by ELISA, and in CNS by real time PCR for IgG RNA. Inflammation, microglial activation, axonal damage and demyelination were assessed using immunohistochemistry. 5D2-induced BCD was confirmed by demonstration of nearly absent CD19(+) cells in the blood and lymphoid tissue. Systemic and CNS antibody responses were suppressed during 5D2 treatment. Higher viral loads were detected in 5D2-treated mice than in controls, and the viral levels correlated negatively with IgG production in the brain. Overall, 5D2 caused worsening of the early encephalitis and faster progression of disability, as well as exacerbation of the pathology of TMEV-IDD at the end stage of the disease. These data indicate that BCD in humans might worsen CNS viral infections and might not improve disability accrual in MS.

The systemic and local acute phase response following acute brain injury.

It is not known whether acute brain injury results in a systemic acute phase response (APR) or whether an APR influences outcome after an insult to the CNS. The present study sought to establish whether brain injury elicits a systemic or local APR. The expression of acute phase protein (APP) mRNA in liver and brain tissues was measured by Taqman reverse transcriptase-polymerase chain reaction after an excitotoxic lesion in the striatum or challenge with a proinflammatory cytokine. N-methyl-d-aspartate (NMDA)-induced brain lesion did not elicit a systemic APR. In contrast, proinflammatory challenge with mouse recombinant interleukin-1beta (mrIL-1beta) resulted in a significant hepatic APP mRNA expression within 6 hours. Thus, an inflammatory challenge that results in a meningitis leads to a hepatic APR, whereas acute brain injury alone, with no evidence of a meningitis, does not produce an APR. This is surprising because NMDA leads to an increase in endogenous IL-1beta synthesis. This suggests that the brain has an endogenous antiinflammatory mechanism, which protects against the spread of inflammation after an acute injury. In the brain, both excitotoxic lesions and proinflammatory challenge resulted in a profound parenchymal upregulation of APP mRNA after 6 and 12 hours in the injected hemisphere. These results suggest that the local APR may play a role as an antiinflammatory mechanism. These findings indicate a potentially pivotal role for peripheral and local APP production on outcome after brain injury.

Study of cytokine induced neuropathology by high resolution proton NMR spectroscopy of rat urine.

Multiple sclerosis is a major cause of non-traumatic neurological disability. The identification of markers that differentiate disease progression is critical to effective therapy. A combination of NMR spectroscopic metabolic profiling of urine and statistical pattern recognition was used to detect focal inflammatory central nervous system (CNS) lesions induced by microinjection of a replication-deficient recombinant adenovirus expressing TNF-alpha or IL1-beta cDNA into the brains of Wistar rats. These animals were compared with a group of naïve rats and a group of animals injected with an equivalent null adenovirus. Urine samples were collected 7 days after adenovirus injection, when the inflammatory lesion is maximally active. Principal components analysis and Partial Least Squares-Discriminate analysis of the urine (1)H NMR spectra revealed significant differences between each of the cytokine adenovirus groups and the control groups; for the TNF-alpha group the main differences lay in citrate and succinate, while for the IL-1beta group the predominant changes occurred in leucine, isoleucine, valine and myo-inositol. Thus, we can identify urinary metabolic vectors that not only separate rats with inflammatory lesions in the brain from control animals, but also distinguish between different types of CNS inflammatory lesions.