Growth Factor Treatment of Demyelinating Disease: at Last, a Leap into the Light

Trends in Immunology. Nov, 2002  |  Pubmed ID: 12401395

Researchers seeking treatments for multiple sclerosis (MS) have long dreamed of using neurotrophic factors to enhance remyelination. Previous attempts to apply trophic support for oligodendrocytes in experimental demyelination uniformly produced complicated outcomes that reflected unexpected effects on immune or inflammatory responses and could be interpreted only with caution. Now, two recent publications have demonstrated convincingly that cytokines of the interleukin (IL)-6 superfamily can ameliorate experimental autoimmune encephalomyelitis and promote oligodendrocyte survival, without demonstrable effect on inflammation or immune responses.

Interleukin-6 Protects Anterior Horn Neurons from Lethal Virus-induced Injury

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2003  |  Pubmed ID: 12533608

We evaluated the role of interleukin-6 (IL-6) in neuronal injury after CNS infection. IL-6-/- and IL-6+/+ mice of resistant major histocompatibility complex (MHC) H-2b haplotype intracerebrally infected with Theiler's virus cleared the infection normally without development of viral persistence, lethal neuronal infection, or late phase demyelination. In contrast, infection of IL-6-/- mice on a susceptible H-2q haplotype resulted in frequent deaths and severe neurologic deficits within 2 weeks of infection as compared with infected IL-6+/+ H-2q littermate controls. Morphologic analysis demonstrated dramatic injury to anterior horn neurons of IL-6-/- H-2q mice at 12 d after infection. Infectious viral titers in the CNS (brain and spinal cord combined) were equivalent between IL-6-/- H-2q and IL-6+/+ H-2q mice. In contrast, more viral RNA was detected in the spinal cord of IL-6-/- mice compared with IL-6+/+ H-2q mice. Virus antigen was localized predominantly to anterior horn cells in infected IL-6-/- H-2q mice. IL-6 deletion did not affect the humoral response directed against virus, nor did it affect the expression of CD4, CD8, MHC class I, or MHC class II in the CNS. Importantly, IL-6 was expressed by astrocytes of infected IL-6+/+ mice but not in astrocytes of IL-6-/- mice or uninfected IL-6+/+ mice. Furthermore, expression of various chemokines was robust at 12 d after infection in both H-2b and H-2q IL-6-/- mice, indicating that intrinsic CNS inflammatory responses did not depend on the presence of IL-6. Finally, in vitro analysis of virus-induced death in neuroblastoma-spinal cord-34 motor neurons and primary anterior horn cell neurons showed that IL-6 exerted a neuroprotective effect. These data support the hypothesis that IL-6 plays a critical role in protecting specific populations of neurons from irreversible injury.

Depolarization of PC12 Cells Induces Neurite Outgrowth and Enhances Nerve Growth Factor-induced Neurite Outgrowth in Rats

Neuroscience Letters. Nov, 2003  |  Pubmed ID: 14550909

Synaptic plasticity is clearly controlled by synaptic activity and by neurotrophin-dependent signaling. We have previously hypothesized that synaptic activity modulates concomitant neurotrophin receptor signaling, thereby integrating the activity state of a synapse with the state of neurotrophic support available at the synapse. Herein we present evidence in support of this hypothesis. Using PC12 cells as a model of the presynaptic element, we show that depolarization increases TrkA tyrosine phosphorylation in response to nerve growth factor (NGF). Moreover, we show that depolarization alone is sufficient to induce the tyrosine phosphorylation of TrkA. These findings are functionally relevant, as evidenced by our observation that depolarization alone induces neurite outgrowth, and that depolarization dramatically enhances neurite outgrowth in response to NGF, especially in primed PC12 cells. We conclude that normal synaptic function may depend upon the integration of synaptic activity and activity-dependent neurotrophin release and signaling, and that these findings have potential relevance to neural repair.

Gamma Interferon is Critical for Neuronal Viral Clearance and Protection in a Susceptible Mouse Strain Following Early Intracranial Theiler's Murine Encephalomyelitis Virus Infection

Journal of Virology. Nov, 2003  |  Pubmed ID: 14581562

We evaluated the role of gamma interferon (IFN-gamma) in protecting neurons from virus-induced injury following central nervous system infection. IFN-gamma(-/-) and IFN-gamma(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-gamma(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-gamma(-/-) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-gamma(-/-) mice of the susceptible H-2(q) haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-gamma(-/-) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-gamma(-/-) H-2(q) mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-gamma(-/-) H-2(q) mice. IFN-gamma deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-gamma(-/-) H-2(q) mice was lower than those in IFN-gamma(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-gamma exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-gamma plays a critical role in protecting spinal cord neurons from persistent infection and death.

Human Monoclonal IgM Antibody Promotes CNS Myelin Repair Independent of Fc Function

Brain Pathology (Zurich, Switzerland). Oct, 2003  |  Pubmed ID: 14655764

The human monoclonal IgM antibody sHIgM22 and mouse IgM monoclonal antibody 94.03 bind to oligodendrocytes, induce calcium signals in cultured glial cells, and promote remyelination in mouse models of multiple sclerosis. In order to address the mechanisms employed by these antibodies to promote CNS repair, bivalent monomers, F(ab')2 fragments, and monovalent forms of these antibodies were investigated to determine whether they exhibit the same remyelinating potential as the intact IgMs. The two antibodies displayed different structural requirements for retention of function. Antibody sHIgM22 remained functional even when reduced to a bivalent F(ab')2 fragment, while disruption of the pentameric structure of antibody 94.03 destroyed its functional properties. Competition studies demonstrated that the two antibodies recognize different entities on the surface of glial cells. These results indicate that the constant region and pentameric structure of IgM is not always necessary for the stimulation of myelin repair, eliminating the requirement for IgM immune effector functions in this process. The ability of the antibodies to cross-link cell surface determinants on oligodendrocytes appears to be an essential aspect of the mechanism of cellular activation. The finding that two antibodies, which induce similar in vivo effects, bind to different structures, and have different cross-linking requirements suggests that activation of glial cells involves the rearrangement of a complex membrane compartment.

Trafficking the NGF Signal: Implications for Normal and Degenerating Neurons

Progress in Brain Research. 2004  |  Pubmed ID: 14699953

Nerve growth factor (NGF) activates TrkA to trigger signaling events that promote the survival, differentiation and maintenance of neurons. The mechanism(s) that controls the retrograde transport of the NGF signal from axon terminals to neuron cell bodies is not known. The 'signaling endosome' hypothesis stipulates that NGF, TrkA and signaling proteins are retrogradely transported on endocytic vesicles. Here, we provide evidence for the existence of signaling endosomes. Following NGF treatment, clathrin-coated vesicles (CCVs) contain NGF bound to TrkA together with activated signaling proteins of the Ras/pErk1/2 pathway. NGF signals from isolated CCVs through the Erk1/2 pathway. Early endosomes appear to represent a second type of signaling endosomes. We found that NGF induced a sustained activation of Rap1, a small monomeric GTP-binding protein of the Ras family, and that this activation occurred in early endosomes that contain key elements of Rap1/pErk1/2 pathway. We discuss the possibility that the failure of retrograde NGF signaling in a mouse model of Down syndrome (Ts65Dn) may be due to the failure to retrograde transport signaling endosomes. It is important to define further the significance of signaling endosomes in the biology of both normal and degenerating neurons.

Signaling Endosome Hypothesis: A Cellular Mechanism for Long Distance Communication

Journal of Neurobiology. Feb, 2004  |  Pubmed ID: 14704953

The kinetics of signaling endosome retrograde transport along axons is analyzed and offered as evidence that such transport is more efficient than diffusion or calcium wave-based signaling systems over even relatively small distances. Evidence is provided to support the signaling endosome hypothesis and to expand the hypothesis to include signaling in many cell types and many cellular dimensions. Finally, a saltatory, regenerating inositol 1,4,5-trisphosphate wave model is offered to reconcile current discrepancies in the literature regarding endosomal-based retrograde signaling.

Antiapoptotic Signaling by a Remyelination-promoting Human Antimyelin Antibody

Neurobiology of Disease. Feb, 2004  |  Pubmed ID: 14751777

Stabilizing the survival of oligodendrocytes and oligodendrocyte precursors within and near lesions in patients suffering from multiple sclerosis (MS) and other demyelinating diseases is an important therapeutic goal. Previous studies have identified a human-derived monoclonal IgM antibody designated rHIgM22 that induces remyelination in a mouse model of MS. We provide evidence that this antibody, directed against myelin, induces antiapoptotic signaling in premyelinating oligodendrocytes and reduces caspase-3 activation and caspase gene expression in mice undergoing antibody-induced remyelination. This effect was dependent on calcium entry via CNQX-sensitive channels and on lipid raft integrity, and was correlated with suppression of JNK signaling. We conclude that rHIgM22 may induce remyelination via rescue of oligodendrocytes, and suggest that such autoantibody-mediated signaling may have important therapeutic implications for a variety of neurological diseases, including stroke and Alzheimer's disease.

Differences in the Surface Binding and Endocytosis of Neurotrophins by P75NTR

Molecular and Cellular Neurosciences. Jun, 2004  |  Pubmed ID: 15207854

Neurotrophins transmit signals retrogradely from synapses to cell bodies by two different types of surface receptors, p75NTR and Trks. Compared to TrkA, the function of p75NTR in nerve growth factor (NGF) endocytosis is less clear, and it is unknown whether p75NTR by itself may internalize other neurotrophins besides NGF. We directly compared TrkA and p75NTR for their ability to internalize NGF, and we also examined the endocytosis of iodinated brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) by p75NTR. Cells expressing solely TrkA internalized NGF more efficiently than cells expressing p75NTR. Surprisingly, cells expressing only p75NTR internalized far more BDNF or NT3 than NGF. Moreover, p75NTR was more important for surface binding than for intracellular accumulation of each neurotrophin. Finally, we established a mechanistic role for the clathrin pathway in p75NTR endocytosis. Our results suggest that p75NTR may have multiple roles in different subcellular locations, functioning both at the cell surface and also within endocytic compartments.

Long-distance Retrograde Neurotrophic Signaling

Current Opinion in Neurobiology. Feb, 2005  |  Pubmed ID: 15721743

The retrograde communication of neurotrophic signals from axon terminals to neuron cell bodies is crucial for neuron survival and plasticity. Several mechanisms have been proposed in the past, but recent evidence strongly supports the hypothesis that the retrograde propagation of self-regenerating signaling organelles, derived from the endocytosis of activated neurotrophin-bound receptor tyrosine kinases, is the primary mechanism responsible for this long-distance communication.

Differential Endocytic Sorting of P75NTR and TrkA in Response to NGF: a Role for Late Endosomes in TrkA Trafficking

Molecular and Cellular Neurosciences. Mar, 2005  |  Pubmed ID: 15737746

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR.

Modeling the Signaling Endosome Hypothesis: Why a Drive to the Nucleus is Better Than a (random) Walk

Theoretical Biology & Medical Modelling. 2005  |  Pubmed ID: 16236165

Information transfer from the plasma membrane to the nucleus is a universal cell biological property. Such information is generally encoded in the form of post-translationally modified protein messengers. Textbook signaling models typically depend upon the diffusion of molecular signals from the site of initiation at the plasma membrane to the site of effector function within the nucleus. However, such models fail to consider several critical constraints placed upon diffusion by the cellular milieu, including the likelihood of signal termination by dephosphorylation. In contrast, signaling associated with retrogradely transported membrane-bounded organelles such as endosomes provides a dephosphorylation-resistant mechanism for the vectorial transmission of molecular signals. We explore the relative efficiencies of signal diffusion versus retrograde transport of signaling endosomes.

Picornaviruses and Cell Death

Trends in Microbiology. Jan, 2006  |  Pubmed ID: 16337385

Members of the picornavirus family, including poliovirus and foot-and-mouth disease virus, are widespread pathogens of humans and domestic animals. Recent global developments in the resurgence of poliovirus infection and in the control of foot-and-mouth disease infection highlight the problems caused by the ability of picornaviruses to alter the apoptotic machinery of host cells and establish persistent infections. Despite the medical, economic and social impact of this family of viruses, little information exists that integrates the mechanisms of cell death and damage induced by related family members. Fortunately, examination of the reported roles and functions of individual viral proteins from multiple picornaviruses makes it possible to surmise canonical functions for these proteins. This review analyzes the canonical function of picornavirus proteins involved in the alteration of apoptotic homeostasis in infected host cells.

Activated Microglia Stimulate Transcriptional Changes in Primary Oligodendrocytes Via IL-1beta

Neurobiology of Disease. Sep, 2006  |  Pubmed ID: 16887357

No therapy currently exists to repair demyelinated lesions in multiple sclerosis. However, the use of IgM antibodies may provide a valuable therapeutic avenue for evoking such repair. Unfortunately, the mechanism of immunoglobulin action in CNS repair is currently unknown but may depend upon complex interactions between multiple cell types rather than upon direct activation of a single cell type. Using rat mixed glial cultures containing oligodendrocytes, microglia, and astrocytes, we found that the Fc portion of human IgM shifts microglia to an activated phenotype, reduces glial proliferation, upregulates a variety of immediate early genes, including JunB, Egr-1, and c-Fos, and stimulates microglial production and release of IL-1beta. Microglia-derived IL-1beta consequently triggers transcriptional upregulation of immediate early genes such as c-Jun, Egr-1, and c-Fos in the mixed glial cultures, and stimulates the upregulation of late response genes such as lipocalin in purified oligodendrocytes. Treatment with an IL-1beta receptor antagonist abrogates the effects of Fcmu on glial proliferation and prevents the upregulation of lipocalin in response to Fcmu, but does not prevent Fcmu-mediated upregulation of IL-1beta, suggesting that IL-1beta mediates at least some of the downstream effects of Fcmu in mixed glial cultures. We hypothesize that Fcmu-stimulated IL-1beta-induced upregulation of immediate early and late response genes in oligodendrocytes may promote CNS repair.

Disrupted Spatial Memory is a Consequence of Picornavirus Infection

Neurobiology of Disease. Nov, 2006  |  Pubmed ID: 16919964

Picornaviruses are a socioeconomically important family of viruses that includes the rhinoviruses and enteroviruses. Many of these viruses, including the "common cold" Coxsackie virus A21, maintain neurovirulent potential and may induce hippocampal injury. The behavioral implications of this injury have not been adequately explored. Using C57BL/6J mice infected with Theiler's murine encephalomyelitis virus, we examined the formation of spatial memories using the Morris water maze test. Virus-infected mice had greater search error compared to sham-infected animals during the location of a hidden platform and were unable to discriminate the location of the training quadrant during the final probe trial. Furthermore, sham-infected mice were place responders whereas virus-infected mice were cue responders, indicating a lack of spatial memory formation in infected animals. Importantly, the degree of memory impairment was correlated to the extent of hippocampal injury. This suggests that picornavirus infection of the human CNS may also result in at least some degree of neurologic deficit. An important implication of such subclinical virus-induced neurologic deficit is that the injury may accumulate over the lifetime of the individual, eventually leading to the manifestation of clinical cognitive or memory deficits.

Induction of a Gene Expression Program in Dendritic Cells with a Cross-linking IgM Antibody to the Co-stimulatory Molecule B7-DC

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Nov, 2006  |  Pubmed ID: 17023390

While bivalent antibodies can block ligand-receptor interactions, IgM pentamers efficiently cross-link cell surface targets and evoke physiological responses. We have described one such interaction between an IgM antibody (Ab) and the B7-DC costimulatory molecule expressed by dendritic cells that induces strong antitumor immunity and modulates pathogenic responses associated with allergic asthma. Progressive changes in gene expression in dendritic cells activated by an IgM B7-DC cross-linking Ab resulted in the increased expression in 350 genes and decreased expression of more than 200 genes over the course of 24 h following Ab treatment. In particular, up-regulation of the caspase inhibitor FLIP and the chemokine receptor CCR7, and the down-regulation of the CXCR4 receptor provide a mechanistic basis of Ab-induced survival and enhanced migration into draining lymph nodes. Increased expression of both cell surface and secreted molecules known to be mediators of the immunomodulatory properties of dendritic cells was detected at both the levels of RNA and protein expression. This analysis documents the ability of IgM Ab to activate a gene expression cascade leading to important biological changes in cellular function and provides mechanistic insight into the potent immunomodulatory properties attributed to this Ab.

Coated Glass and Vicryl Microfibers As Artificial Axons

Cells, Tissues, Organs. 2006  |  Pubmed ID: 17159344

The complex interactions that occur between oligodendrocytes and axons during the process of central nervous system myelination and remyelination remain unclear. Elucidation of the cell-biological and -biochemical mechanisms supporting myelin production and elaboration requires a robust in vitro system that recapitulates the relationship between axons and oligodendrocytes in a manner that is open to molecular dissection. We provide evidence for an artificial axon culture system in which we observed oligodendrocytes extending large plasma membrane projections that frequently completely ensheathed fibers coated with a variety of extracellular matrix molecules. These membrane projections varied in extent and thickness depending upon the substrate and upon the diameter of the coated fiber. Matrigel-coated glass microfibers were found to support the development of thick membrane sheaths that extended for hundreds of microns and exhibited many features suggestive of the potential for true myelin deposition. Likewise, Matrigel-coated Vicryl fibers supported plasma membrane extensions that covered extremely large surface areas and occasionally wrapped the coated Vicryl fibers in more than one membrane layer. Our findings suggest that the deposition of molecular cues onto glass or polymer fibers either via adsorption or chemical modification may be a useful tool for the discovery or validation of axonal factors critical for myelination and remyelination. Herein, we provide evidence that polyglactin 910 and glass microfibers coated with adhesion factors may provide a reasonable system for the in vitro analysis of myelination, and may eventually serve a role in engineering artificial systems for neural repair.

Searching Historical Herbal Texts for Potential New Drugs

BMJ (Clinical Research Ed.). Dec, 2006  |  Pubmed ID: 17185721

A High-throughput 3-parameter Flow Cytometry-based Cell Death Assay

Cytometry. Part A : the Journal of the International Society for Analytical Cytology. Mar, 2007  |  Pubmed ID: 17226860

Apoptosis plays a role in many disease states, and the evaluation of novel therapeutics that alters the apoptotic cascade is an area of intense investigation. However, many generally available methods to evaluate cell death are either time consuming, imprecise, or both. We report a system that permits simultaneous evaluation of three apoptotic markers (cell membrane integrity, mitochondrial membrane potential, and cell cycle progression) with minimal technical manipulation. This system is particularly well-suited for toxicologic evaluation of novel compounds and profiling of new apoptosis-inducing agents.

Mechanisms of Action of IVIG in Adult Onset Rasmussen's Encephalitis

The Canadian Journal of Neurological Sciences. Le Journal Canadien Des Sciences Neurologiques. Feb, 2007  |  Pubmed ID: 17352358

Beta-methylamino-alanine (BMAA) Injures Hippocampal Neurons in Vivo

Neurotoxicology. May, 2007  |  Pubmed ID: 17379313

The unusually high incidence of amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC) among the Chamorro people of Guam has fueled an intense search for the etiologic agent responsible for this neurodegenerative disease. Recently, a biomagnification hypothesis was proposed to account for the role of dietary consumption of beta-methylamino-alanine (BMAA) in patients with ALS/PDC. However, this hypothesis is hotly debated and a direct association between BMAA and neuronal injury in vivo has been lacking. We provide evidence that introduction of BMAA into the CNS of mice leads to sporadic death of hippocampal neurons, supporting a direct causal link between BMAA and neuronal injury.

CD8+ T Cells Directed Against a Viral Peptide Contribute to Loss of Motor Function by Disrupting Axonal Transport in a Viral Model of Fulminant Demyelination

Journal of Neuroimmunology. Aug, 2007  |  Pubmed ID: 17493690

Demyelination, a pathological hallmark of multiple sclerosis, may be a necessary but not a sufficient condition for motor dysfunction associated with this disease. We favor a neurodegenerative model of multiple sclerosis and suggest that demyelination creates a permissive environment wherein the denuded axon becomes susceptible to immune-mediated injury. Unfortunately, the cellular effectors responsible for eliciting such axonal injury are currently unknown. Based on previous observations implicating cytotoxic T cells in this injury, we assessed motor function, axon dropout, and axon injury following peptide depletion of the immunodominant CD8+ antiviral T cell response in the IFNgamma receptor-deficient mouse model of acute demyelination. We found that the targeted removal of this population of cytotoxic effector cells prior to infection with the Theiler's murine encephalomyelitis virus caused a substantial preservation of motor function at 45 days postinfection that was associated with preservation of retrograde axonal transport in a subpopulation of surviving axons within the spinal cord. We conclude that cytotoxic T cells may be responsible for the initiation of axon injury following demyelination.

A Randomized Phase I Study of Atuna Racemosa: a Potential New Anti-MRSA Natural Product Extract

Journal of Ethnopharmacology. Dec, 2007  |  Pubmed ID: 17889468

We previously reported significant antimicrobial activity against Gram-positive bacteria from the extract of the Atun tree (Atuna racemosa), identified through rapid digital bioprospecting of a 400-year-old historic herbal text. Toxicity studies in human cell lines showing safety, combined with the ethnomedical descriptions of botanical use, suggested that this extract might be clinically useful against topical Gram-positive bacteria infections.

Absence of Perforin Expression Confers Axonal Protection Despite Demyelination

Neurobiology of Disease. Feb, 2007  |  Pubmed ID: 17112732

Current evidence suggests that demyelination may be a necessary but not a sufficient condition for neurologic deficits associated with multiple sclerosis. Axon injury that occurs within the permissive environment of the demyelinated lesion is better correlated with functional deficits, but the mechanisms and cellular effectors of this injury are largely unknown. In an effort to identify potential axon injury mediators, we examined demyelination, motor function, and the number of spinal axons in perforin-deficient mice. Perforin is a critical molecular mediator of cytotoxic immunological injury and we hypothesized that genetic deletion of perforin expression would protect demyelinated axons. Indeed, we found that while perforin-deficient mice had considerable spinal cord demyelination 180 days after infection with Theiler's murine encephalomyelitis virus, such mice exhibited functional and axonal preservation comparable to non-demyelinated perforin-competent controls. We conclude that perforin-dependent effector cells such as cytotoxic T cells, gammadelta T cells, and natural killer cells may play a role in axon damage that is dependent upon but separable from demyelination.

TRAIL Mediates Liver Injury by the Innate Immune System in the Bile Duct-ligated Mouse

Hepatology (Baltimore, Md.). Apr, 2008  |  Pubmed ID: 18220275

The contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand expressed by cells of the innate immune system, to cholestatic liver injury has not been explored. Our aim was to ascertain if TRAIL contributes to liver injury in the bile duct-ligated (BDL) mouse. C57/BL6 wild-type (wt), TRAIL heterozygote (TRAIL(+/-)), and TRAIL knockout (TRAIL(-/-)) mice were used for these studies. Liver injury and fibrosis were examined 7 and 14 days after BDL, respectively. Hepatic TRAIL messenger RNA (mRNA) was 6-fold greater in BDL animals versus sham-operated wt animals (P < 0.01). The increased hepatic TRAIL expression was accompanied by an increase in liver accumulation of natural killer 1.1 (NK 1.1)-positive NK and natural killer T (NKT) cells, the predominant cell types expressing TRAIL. Depletion of NK 1.1-positive cells reduced hepatic TRAIL mRNA expression and serum alanine aminotransferase (ALT) values. Consistent with a role for NK/NKT cells in this model of liver injury, stress ligands necessary for their recognition of target cells were also up-regulated in hepatocytes following BDL. Compared to sham-operated wt mice, BDL mice displayed a 13-fold increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and an 11-fold increase in caspase 3/7-positive hepatocytes (P < 0.01). The number of TUNEL and caspase 3/7-positive cells was reduced by >80% in BDL TRAIL knockout animals (P < 0.05). Likewise, liver histology, number of bile infarcts, serum ALT values, hepatic fibrosis, and animal survival were also improved in BDL TRAIL(-/-) animals as compared to wt animals. Conclusion: These observations support a pivotal role for TRAIL in cholestatic liver injury mediated by NK 1.1-positive NK/NKT cells.

Human HLA-DR Transgenes Protect Mice from Fatal Virus-induced Encephalomyelitis and Chronic Demyelination

Journal of Virology. Apr, 2008  |  Pubmed ID: 18234804

We evaluated the participatory role of human HLA-DR molecules in control of virus from the central nervous system and in the development of subsequent spinal cord demyelination. The experiments utilized intracranial infection with Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that, in some strains of mice, results in primary demyelination. We studied DR2 and DR3 transgenic mice that were bred onto a combined class I-deficient mouse (beta-2 microglobulin deficient; beta2m(0)) and class II-deficient mouse (Abeta(0)) of the H-2(b) background. Abeta(0).beta2m(0) mice infected with TMEV died within 18 days of infection. These mice showed severe encephalomyelitis due to rapid replication of virus genome. In contrast, transgenic mice with insertion of a single human class II major histocompatibility complex (MHC) gene (DR2 or DR3) survived the acute infection. DR2 and DR3 mice controlled virus infection by 45 days and did not develop spinal cord demyelination. Levels of virus RNA were reduced in HLA-DR transgenic mice compared to Abeta(0).beta2m(0) mice. Virus-neutralizing antibody responses did not explain why DR mice survived the infection and controlled virus replication. However, DR mice showed an increase in gamma interferon and interleukin-2 transcripts in the brain, which were associated with protection. The findings support the hypothesis that the expression of a single human class II MHC molecule can, by itself, influence the control of an intracerebral pathogen in a host without a competent class I MHC immune response. The mechanism of protection appears to be the result of cytokines released by CD4(+) T cells.

NKG2D Contributes to Efficient Clearance of Picornavirus from the Acutely Infected Murine Brain

Journal of Neurovirology. May, 2008  |  Pubmed ID: 18569460

Activated murine cytotoxic T cells express the NKG2D natural cytotoxicity receptor. This receptor recognizes major histocompatibility complex (MHC) class I-like molecules expressed on the surface of infected cells and serves to augment T cell-mediated cytotoxicity. The role of NKG2D-mediated augmentation in the clearance of central nervous system viral infections has not been explored. Using the Theiler's murine encephalomyelitis virus model, the authors found that NKG2D-positive CD8+ cytotoxic T cells enter the brain, that NKG2D ligands are expressed in the brain during acute infection, and that interruption of NKG2D ligand recognition via treatment with a function-blocking antibody attenuates the efficacy of viral clearance from the central nervous system.

Aquaporin-4-binding Autoantibodies in Patients with Neuromyelitis Optica Impair Glutamate Transport by Down-regulating EAAT2

The Journal of Experimental Medicine. Oct, 2008  |  Pubmed ID: 18838545

Neuromyelitis optica (NMO)-immunoglobulin G (IgG) is a clinically validated serum biomarker that distinguishes relapsing central nervous system (CNS) inflammatory demyelinating disorders related to NMO from multiple sclerosis. This autoantibody targets astrocytic aquaporin-4 (AQP4) water channels. Clinical, radiological, and immunopathological data suggest that NMO-IgG might be pathogenic. Characteristic CNS lesions exhibit selective depletion of AQP4, with and without associated myelin loss; focal vasculocentric deposits of IgG, IgM, and complement; prominent edema; and inflammation. The effect of NMO-IgG on astrocytes has not been studied. In this study, we demonstrate that exposure to NMO patient serum and active complement compromises the membrane integrity of CNS-derived astrocytes. Without complement, astrocytic membranes remain intact, but AQP4 is endocytosed with concomitant loss of Na(+)-dependent glutamate transport and loss of the excitatory amino acid transporter 2 (EAAT2) . Our data suggest that EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex. Transport-competent EAAT2 protein is up-regulated in differentiating astrocyte progenitors and in nonneural cells expressing AQP4 transgenically. Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data. Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

TREM-2 Mediated Signaling Induces Antigen Uptake and Retention in Mature Myeloid Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2008  |  Pubmed ID: 19017976

Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-gamma1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.

Functional Characterization of Mouse Spinal Cord Infiltrating CD8+ Lymphocytes

Journal of Neuroimmunology. Sep, 2009  |  Pubmed ID: 19596449

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler's virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8(+) lymphocytes that are CD45(hi)CD44(lo)CD62L(-) and a population of spinal cord infiltrating target effector memory CD8(+) lymphocytes that are CD45(hi)CD44(hi)CD62L(-). These cells respond robustly to ex vivo stimulation by producing interferon gamma but do not exhibit specificity for Theiler's virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities.

Apoptosis of Hippocampal Pyramidal Neurons is Virus Independent in a Mouse Model of Acute Neurovirulent Picornavirus Infection

The American Journal of Pathology. Aug, 2009  |  Pubmed ID: 19608874

Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theiler's murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non-cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection.

Preparation of Biologically Active Subcellular Fractions Using the Balch Homogenizer

Analytical Biochemistry. Nov, 2009  |  Pubmed ID: 19622341

Obtaining vesicular fractions from cell lines or animal tissue is both time and technically intensive. The presence of plasma membrane and nuclear contaminants within a preparation is often dependent on the method of homogenization and is usually mitigated through the use of density gradients. We have developed a method that utilizes Balch homogenization and differential centrifugation to obtain two distinct vesicular fractions along with purified nuclear, cytoplasmic, and ghost fractions within a 3-h period of time without the use of density gradients. Importantly, these fractions maintain their biologic activity following isolation and may be used for both localization and biochemical analyses.

Demyelinated Axons and Motor Function Are Protected by Genetic Deletion of Perforin in a Mouse Model of Multiple Sclerosis

Journal of Neuropathology and Experimental Neurology. Sep, 2009  |  Pubmed ID: 19680139

Axon injury is a major determinant of the loss of neurological function in patients with multiple sclerosis. It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8 T cells and perforin in the Theiler murine encephalomyelitis virus model of multiple sclerosis have used mouse strains resistant to Theiler murine encephalomyelitis virus infection. To test the role of CD8 T cells in axon injury, we established a perforin-deficient mouse model on the H-2 major histocompatibility complex background thereby removing confounding factors related to viral biology in this Theiler murine encephalomyelitis virus-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2 mice, but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediator of axon injury and lend additional support to the hypothesis that CD8 T cells are primarily responsible for axon damage in multiple sclerosis.

Subcutaneous IGF-1 is Not Beneficial in 2-year ALS Trial

Neurology. Oct, 2009  |  Pubmed ID: 19822878

Inflammatory Changes in the Central Nervous System Are Associated with Behavioral Impairment in Plasmodium Berghei (strain ANKA)-infected Mice

Experimental Parasitology. Jul, 2010  |  Pubmed ID: 20138873

Experimental cerebral malaria is a neuroinflammatory condition that results from the host immune response to the parasite. Using intravital microscopy, we investigated leukocyte recruitment in the brain microcirculation and the temporal relationship of this process to the behavioral changes observed in Plasmodium berghei (strain ANKA)-infected C57Bl/6 mice. We found that leukocyte recruitment was increased from day 5 post-infection (p.i.) onwards. Histopathological changes and increased levels of inflammatory cytokines in the brain were also observed. Behavioral performance evaluated by the SHIRPA protocol showed functional impairment from day 6 p.i. onwards. Thus, early leukocyte migration into the brain and associated inflammatory changes may be involved in neurological impairment in parasite-infected C57Bl/6 mice.

Retraction: TREM-2 Mediated Signaling Induces Antigen Uptake and Retention in Mature Myeloid Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2010  |  Pubmed ID: 20483797

CD8+ T Cells Cause Disability and Axon Loss in a Mouse Model of Multiple Sclerosis

PloS One. 2010  |  Pubmed ID: 20814579

The objective of this study was to test the hypothesis that CD8+ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8+ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice.

Therapeutic Doses of Cranial Irradiation Induce Hippocampus-dependent Cognitive Deficits in Young Mice

Journal of Neuro-oncology. Nov, 2011  |  Pubmed ID: 21499912

Fractionated cranial irradiation is an essential part of treatment in the management of cohorts of pediatric brain tumor and leukemia patients. Ionizing radiation damages normal brain parenchyma through a variety of poorly understood mechanisms and results in cognitive dysfunction and significant life-long disability. The goal of our study was to establish and characterize a mouse model of radiation-induced damage to the developing nervous system. Male C57BL/6 mice were exposed to a total dose of 20 Gy of fractionated cranial irradiation at 1 month of age to assess the early and late effects of clinically relevant irradiation doses on the young mouse brain. Compared to age-matched controls, an acute and prolonged decrease in proliferation and the number of immature neurons in the stem cell niche of the hippocampal subgranular zone within the dentate gyrus at 72 h and 1 month following cranial irradiation was noted. Behavioral characterization at 1 and 5 months post-radiation demonstrated significant, persistent and progressive hippocampus-dependent learning deficits. Our study characterizes a clinically relevant mouse model of radiation-induced damage that serves as a platform for future evaluation of therapeutic interventions that may mitigate such cognitive damage. Our research also emphasizes the need for targeted treatment strategies that protect regions of neurogenesis while maximizing therapeutic effects in pediatric cancer patients.

The STAT3 Beacon: IL-6 Recurrently Activates STAT 3 from Endosomal Structures

Experimental Cell Research. Aug, 2011  |  Pubmed ID: 21619877

Endocytic trafficking plays an important role in signal transduction. Signal transducer and activator of transcription 3 (STAT3) and mitogen-activate protein kinase (MAPK) have both been localized to endosomal structures and are dependent upon endocytosis for downstream function. While the dependence of MAPK signaling upon endosomes has been well characterized, the involvement of endosomes in regulating STAT3 signaling has not been defined. Consequently, this study evaluated the role of endosomes in the initiation, modulation, amplification and persistence of interleukin-6(IL-6)-induced STAT3 signal transduction and transcription, and utilized IL-6-induced MAPK signaling as a comparator. Using pharmacologic treatment and temperature control of endocytic trafficking, pulse-chase treatments and in vitro kinase assays, STAT3 was found to interact with endosomes in a markedly different fashion than MAPK. STAT3 was activated by direct interaction with internal structures upstream of the late endosome following IL-6 exposure and persistent STAT3 signaling depended upon recurrent activation from endocytic structures. Further, STAT3 subcellular localization was not dependent upon endocytic trafficking. Instead, STAT3 transiently interacted with endosomes and relocated to the nucleus by an endosome-independent mechanism. Finally, endocytic trafficking played a central role in regulating STAT3 serine 727 phosphorylation through crosstalk with the MAPK signaling system. Together, these data reveal endosomes as central to the genesis, course and outcome of STAT3 signal transduction and transcription.