Drug-resistant seizures associated with hyperinflammatory monocytes in FIRES.

OBJECTIVE: Therapeutic strategies for patients with febrile infection-related epilepsy syndrome (FIRES) are limited, ad hoc, and frequently ineffective. Based on evidence that inflammation drives pathogenesis in FIRES, we used ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) to characterize the monocytic response profile before and after therapy in a child successfully treated with dexamethasone delivered intrathecally six times between hospital Day 23 and 40 at 0.25 mg/kg/dose. METHODS: PBMCs were isolated from serial blood draws acquired during refractory status epilepticus (RSE) and following resolution associated with intrathecal dexamethasone therapy in a previously healthy 9-year-old male that presented with seizures following Streptococcal pharyngitis. Cells were stimulated with bacterial or viral ligands and cytokine release was measured and compared to responses in age-matched healthy control PBMCs. Levels of inflammatory factors in the blood and CSF were also measured and compared to pediatric healthy control ranges. RESULTS: During RSE, serum levels of IL6, CXCL8, HMGB1, S100A8/A9, and CRP were significantly elevated. IL6 was elevated in CSF. Ex vivo stimulation of PBMCs collected during RSE revealed hyperinflammatory release of IL6 and CXCL8 in response to bacterial stimulation. Following intrathecal dexamethasone, RSE resolved, inflammatory levels normalized in serum and CSF, and the PBMC hyperinflammatory response renormalized. SIGNIFICANCE: FIRES may be associated with a hyperinflammatory monocytic response to normally banal bacterial pathogens. This hyperinflammatory response may induce a profound neutrophil burden and the consequent release of factors that further exacerbate inflammation and drive neuroinflammation. Intrathecal dexamethasone may resolve RSE by resetting this inflammatory feedback loop.

Rejuvenation of the aged brain immune cell landscape in mice through p16-positive senescent cell clearance.

Cellular senescence is a plausible mediator of inflammation-related tissue dysfunction. In the aged brain, senescent cell identities and the mechanisms by which they exert adverse influence are unclear. Here we used high-dimensional molecular profiling, coupled with mechanistic experiments, to study the properties of senescent cells in the aged mouse brain. We show that senescence and inflammatory expression profiles increase with age and are brain region- and sex-specific. p16-positive myeloid cells exhibiting senescent and disease-associated activation signatures, including upregulation of chemoattractant factors, accumulate in the aged mouse brain. Senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Activated resident and infiltrating immune cells increase in the aged brain and are partially restored to youthful levels through p16-positive senescent cell clearance in female p16-InkAttac mice, which is associated with preservation of cognitive function. Our study reveals dynamic remodeling of the brain immune cell landscape in aging and suggests senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.

Teriflunomide shifts the astrocytic bioenergetic profile from oxidative metabolism to glycolysis and attenuates TNFα-induced inflammatory responses.

Astrocytes utilize both glycolytic and mitochondrial pathways to power cellular processes that are vital to maintaining normal CNS functions. These cells also mount inflammatory and acute phase reactive programs in response to diverse stimuli. While the metabolic functions of astrocytes under homeostatic conditions are well-studied, the role of cellular bioenergetics in astrocyte reactivity is poorly understood. Teriflunomide exerts immunomodulatory effects in diseases such as multiple sclerosis by metabolically reprogramming lymphocytes and myeloid cells. We hypothesized that teriflunomide would constrain astrocytic inflammatory responses. Purified murine astrocytes were grown under serum-free conditions to prevent acquisition of a spontaneous reactive state. Stimulation with TNFα activated NFκB and increased secretion of Lcn2. TNFα stimulation increased basal respiration, maximal respiration, and ATP production in astrocytes, as assessed by oxygen consumption rate. TNFα also increased glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate, as assessed by measuring the extracellular acidification rate. TNFα specifically increased mitochondrial ATP production and secretion of Lcn2 required ATP generated by oxidative phosphorylation. Inhibition of dihydroorotate dehydrogenase via teriflunomide transiently increased both oxidative phosphorylation and glycolysis in quiescent astrocytes, but only the increased glycolytic ATP production was sustained over time, resulting in a bias away from mitochondrial ATP production even at doses down to 1 µM. Preconditioning with teriflunomide prevented the TNFα-induced skew toward oxidative phosphorylation, reduced mitochondrial ATP production, and reduced astrocytic inflammatory responses, suggesting that this drug may limit neuroinflammation by acting as a metabolomodulator.

Microdialysis and microperfusion electrodes in neurologic disease monitoring.

Contemporary biomarker collection techniques in blood and cerebrospinal fluid have to date offered only modest clinical insights into neurologic diseases such as epilepsy and glioma. Conversely, the collection of human electroencephalography (EEG) data has long been the standard of care in these patients, enabling individualized insights for therapy and revealing fundamental principles of human neurophysiology. Increasing interest exists in simultaneously measuring neurochemical biomarkers and electrophysiological data to enhance our understanding of human disease mechanisms. This review compares microdialysis, microperfusion, and implanted EEG probe architectures and performance parameters. Invasive consequences of probe implantation are also investigated along with the functional impact of biofouling. Finally, previously developed microdialysis electrodes and microperfusion electrodes are reviewed in preclinical and clinical settings. Critically, current and precedent microdialysis and microperfusion probes lack the ability to collect neurochemical data that is spatially and temporally coincident with EEG data derived from depth electrodes. This ultimately limits diagnostic and therapeutic progress in epilepsy and glioma research. However, this gap also provides a unique opportunity to create a dual-sensing technology that will provide unprecedented insights into the pathogenic mechanisms of human neurologic disease.

Citrullinated myelin induces microglial TNFα and inhibits endogenous repair in the cuprizone model of demyelination.

BACKGROUND: Microglia are the primary phagocytes of the central nervous system and are responsible for removing damaged myelin following demyelination. Previous investigations exploring the consequences of myelin phagocytosis on microglial activation overlooked the biochemical modifications present on myelin debris. Such modifications, including citrullination, are increased within the inflammatory environment of multiple sclerosis lesions. METHODS: Mouse cortical myelin isolated by ultracentrifugation was citrullinated ex vivo by incubation with the calcium-dependent peptidyl arginine deiminase PAD2. Demyelination was induced by 6 weeks of cuprizone (0.3%) treatment and spontaneous repair was initiated by reversion to normal chow. Citrullinated or unmodified myelin was injected into the primary motor cortex above the cingulum bundle at the time of reversion to normal chow and the consequent impact on remyelination was assessed by measuring the surface area of myelin basic protein-positive fibers in the cortex 3 weeks later. Microglial responses to myelin were characterized by measuring cytokine release, assessing flow cytometric markers of microglial activation, and RNAseq profiling of transcriptional changes. RESULTS: Citrullinated myelin induced a unique microglial response marked by increased tumor necrosis factor α (TNFα) production both in vitro and in vivo. This response was not induced by unmodified myelin. Injection of citrullinated myelin but not unmodified myelin into the cortex of cuprizone-demyelinated mice significantly inhibited spontaneous remyelination. Antibody-mediated neutralization of TNFα blocked this effect and restored remyelination to normal levels. CONCLUSIONS: These findings highlight the role of post-translation modifications such as citrullination in the determination of microglial activation in response to myelin during demyelination. The inhibition of endogenous repair induced by citrullinated myelin and the reversal of this effect by neutralization of TNFα may have implications for therapeutic approaches to patients with inflammatory demyelinating disorders.

Methods for intratumoral microdialysis probe targeting and validation in murine brain tumor models.

BACKGROUND: Microdialysis is a well validated sampling technique that can be used for pharmacokinetic studies of oncological drugs targeting the central nervous system. This technique has also been applied to evaluate tumor metabolism and identify pharmacodynamic biomarkers of drug activity. Despite the potential utility of microdialysis for therapeutic discovery, variability in tumor size and location hamper routine use of microdialysis as a preclinical tool. Quantitative validation of microdialysis membrane location relative to radiographically evident tumor regions could facilitate rigorous preclinical studies. However, a widely accessible standardized workflow for preclinical catheter placement and validation is needed. NEW METHOD: We provide methods for a workflow to yield tailored placement of microdialysis probes within a murine intracranial tumor and illustrate in an IDH1-mutant patient-derived xenograft (PDX) model. This detailed workflow uses a freely available on-line tool built within 3D-slicer freeware to target microdialysis probe placement within the tumor core and validate probe placement fully within the tumor. RESULTS: We illustrate use of this workflow to validate microdialysis probe location relative to implanted IDH1-mutant PDXs, using the microdialysis probes to quantify levels of extracellular onco-metabolite D-2 hydroxyglutarate. COMPARISON WITH EXISTING METHODS: Previous methods have used 3D slicer to reliably measure tumor volumes. Prior microdialysis studies have targeted expected tumor locations without validation. CONCLUSIONS: The new method offers a streamlined and freely available workflow in 3D slicer to optimize and validate microdialysis probe placement within a murine brain tumor.

Remyelination-Promoting DNA Aptamer Conjugate Myaptavin-3064 Binds to Adult Oligodendrocytes In Vitro.

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

Expanded Clinical Phenotype, Oncological Associations, and Immunopathologic Insights of Paraneoplastic Kelch-like Protein-11 Encephalitis.

Importance: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management. Objective: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis. Design, Setting, and Participants: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays). Main Outcomes and Measures: Outcome variables included modified Rankin score and gait aid use. Results: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell-predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis. Conclusions and Relevance: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.

IgM Natural Autoantibodies in Physiology and the Treatment of Disease.

Antibodies are vital components of the adaptive immune system for the recognition and response to foreign antigens. However, some antibodies recognize self-antigens in healthy individuals. These autoreactive antibodies may modulate innate immune functions. IgM natural autoantibodies (IgM-NAAs) are a class of primarily polyreactive immunoglobulins encoded by germline V-gene segments which exhibit low affinity but broad specificity to both foreign and self-antigens. Historically, these autoantibodies were closely associated with autoimmune disease. Nevertheless, not all human autoantibodies are pathogenic and compelling evidence indicates that IgM-NAAs may exert a spectrum of effects from injurious to protective depending upon cellular and molecular context. In this chapter, we review the current state of knowledge regarding the potential physiological and therapeutic roles of IgM-NAAs in different disease conditions such as atherosclerosis, cancer, and autoimmune disease. We also describe the discovery of two reparative IgM-NAAs by our laboratory and delineate their proposed mechanisms of action in central nervous system (CNS) disease.

Fueling the FIRES: Hemophagocytic lymphohistiocytosis in febrile infection-related epilepsy syndrome.

OBJECTIVES: Although secondary hemophagocytic lymphohistiocytosis (HLH) has been reported in children with critical illness of various etiologies, it has not been reported in patients with febrile infection-related epilepsy syndrome (FIRES). We describe a series of patients with concurrent HLH and FIRES in an effort to establish common pathophysiologic abnormalities. METHODS: Five patients with FIRES who were assessed for HLH were identified from a neurocritical care database. All were previously healthy and had extensive diagnostic testing. All had clinical deterioration with multiorgan dysfunction prompting HLH screening 20-29 days after hospitalization. Markers for inflammatory dysregulation were assessed in cerebrospinal fluid (CSF) and serum at various time points. Outcomes were assessed 6 months after presentation. RESULTS: Three patients met clinical criteria for secondary HLH. Elevation of specific cytokines/chemokines was variable. CSF neopterin, high mobility group box 1 (HMGB1), and C-X-C motif chemokine ligand 8 (CXCL8) were significantly elevated in all. Interleukin-1ß (IL-1ß) and IL-18 were not elevated in any of the samples. Treatment and outcomes were variable. SIGNIFICANCE: We describe 3 patients with HLH and FIRES. The co-occurrence of these 2 rare disorders suggests the possibility of a common immune dysregulation phenotype prolonging epileptogenesis. HLH screening in critically ill patients with FIRES may yield a broader understanding of shared inflammatory processes.

Inflammatory cytokine-induced changes in neural network activity measured by waveform analysis of high-content calcium imaging in murine cortical neurons.

During acute neuroinflammation, increased levels of cytokines within the brain may contribute to synaptic reorganization that results in long-term changes in network hyperexcitability. Indeed, inflammatory cytokines are implicated in synaptic dysfunction in epilepsy and in an array of degenerative and autoimmune diseases of the central nervous system. Current tools for studying the impact of inflammatory factors on neural networks are either insufficiently fast and sensitive or require complicated and costly experimental rigs. Calcium imaging offers a reasonable surrogate for direct measurement of neuronal network activity, but traditional imaging paradigms are confounded by cellular heterogeneity and cannot readily distinguish between glial and neuronal calcium transients. While the establishment of pure neuron cultures is possible, the removal of glial cells ignores physiologically relevant cell-cell interactions that may be critical for circuit level disruptions induced by inflammatory factors. To overcome these issues, we provide techniques and algorithms for image processing and waveform feature extraction using automated analysis of spontaneous and evoked calcium transients in primary murine cortical neuron cultures transduced with an adeno-associated viral vector driving the GCaMP6f reporter behind a synapsin promoter. Using this system, we provide evidence of network perturbations induced by the inflammatory cytokines TNFα, IL1ß, and IFNγ.

Neuroprotection mediated by inhibition of calpain during acute viral encephalitis.

Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel's strain of Theiler's murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders.

NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG.

BACKGROUND: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO. METHODS: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining. RESULTS: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957. CONCLUSIONS: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

Hippocampal protection in mice with an attenuated inflammatory monocyte response to acute CNS picornavirus infection.

Neuronal injury during acute viral infection of the brain is associated with the development of persistent cognitive deficits and seizures in humans. In C57BL/6 mice acutely infected with the Theiler's murine encephalomyelitis virus, hippocampal CA1 neurons are injured by a rapid innate immune response, resulting in profound memory deficits. In contrast, infected SJL and B6xSJL F1 hybrid mice exhibit essentially complete hippocampal and memory preservation. Analysis of brain-infiltrating leukocytes revealed that SJL mice mount a sharply attenuated inflammatory monocyte response as compared to B6 mice. Bone marrow transplantation experiments isolated the attenuation to the SJL immune system. Adoptive transfer of B6 inflammatory monocytes into acutely infected B6xSJL hosts converted these mice to a hippocampal damage phenotype and induced a cognitive deficit marked by failure to recognize a novel object. These findings show that inflammatory monocytes are the critical cellular mediator of hippocampal injury during acute picornavirus infection of the brain.

The STAT3 beacon: IL-6 recurrently activates STAT 3 from endosomal structures.

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.

Therapeutic doses of cranial irradiation induce hippocampus-dependent cognitive deficits in young mice.

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.

Inflammatory changes in the central nervous system are associated with behavioral impairment in Plasmodium berghei (strain ANKA)-infected mice.

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.

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes.

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.

TREM-2 mediated signaling induces antigen uptake and retention in mature myeloid dendritic cells.

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.

TRAIL mediates liver injury by the innate immune system in the bile duct-ligated mouse.

UNLABELLED: 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.