CD8+ T cells recognizing a neuron-restricted antigen injure axons in a model of multiple sclerosis.

CD8+ T cells outnumber CD4+ cells in multiple sclerosis (MS) lesions associated with disease progression, but the pathogenic role and antigenic targets of these clonally expanded effectors are unknown. Based on evidence that demyelination is necessary but not sufficient for disease progression in MS, we previously hypothesized that CNS-infiltrating CD8+ T cells specific for neuronal antigens directly drive the axonal and neuronal injury that leads to cumulative neurologic disability in patients with MS. We now show that demyelination induced expression of MHC class I on neurons and axons and resulted in presentation of a neuron-specific neoantigen (synapsin promoter-driven chicken ovalbumin) to antigen-specific CD8+ T cells (anti-ovalbumin OT-I TCR-transgenic T cells). These neuroantigen-specific effectors surveilled the CNS in the absence of demyelination but were not retained. However, upon induction of demyelination via cuprizone intoxication, neuroantigen-specific CD8+ T cells proliferated, accumulated in the CNS, and damaged neoantigen-expressing neurons and axons. We further report elevated neuronal expression of MHC class I and ß2-microglobulin transcripts and protein in gray matter and white matter tracts in tissue from patients with MS. These findings support a pathogenic role for autoreactive anti-axonal and anti-neuronal CD8+ T cells in MS progression.

G9a Modulates Lipid Metabolism in CD4 T Cells to Regulate Intestinal Inflammation.

BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Preservation of antigen-specific responses in cryopreserved CD4+ and CD8+ T cells expanded with IL-2 and IL-7.

Objectives: We sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens. Methods: Dendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24-72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs, cryopreserved T cells, and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing, 2) expansion, and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs, we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4. Results: We observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However, polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore, resting these expanded T cells in IL-2, IL-7 or IL-15 did not restore the antigen dependent responses. In contrast, T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing. Significance: While screening techniques for potential pathological autoantibodies have come a long way, comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune diseases.

ISGylation is induced in neurons by demyelination driving ISG15-dependent microglial activation.

The causes of grey matter pathology and diffuse neuron injury in MS remain incompletely understood. Axonal stress signals arising from white matter lesions has been suggested to play a role in initiating this diffuse grey matter pathology. Therefore, to identify the most upstream transcriptional responses in neurons arising from demyelinated axons, we analyzed the transcriptome of actively translating neuronal transcripts in mouse models of demyelinating disease. Among the most upregulated genes, we identified transcripts associated with the ISGylation pathway. ISGylation refers to the covalent attachment of the ubiquitin-like molecule interferon stimulated gene (ISG) 15 to lysine residues on substrates targeted by E1 ISG15-activating enzyme, E2 ISG15-conjugating enzymes and E3 ISG15-protein ligases. We further confirmed that ISG15 expression is increased in MS cortical and deep gray matter. Upon investigating the functional impact of neuronal ISG15 upregulation, we noted that ISG15 expression was associated changes in neuronal extracellular vesicle protein and miRNA cargo. Specifically, extracellular vesicle-associated miRNAs were skewed toward increased frequency of proinflammatory and neurotoxic miRNAs and decreased frequency of anti-inflammatory and neuroprotective miRNAs. Furthermore, we found that ISG15 directly activated microglia in a CD11b-dependent manner and that microglial activation was potentiated by treatment with EVs from neurons expressing ISG15. Further study of the role of ISG15 and ISGylation in neurons in MS and neurodegenerative diseases is warranted.

Septin-5 and -7-IgGs: Neurologic, Serologic, and Pathophysiologic Characteristics.

BACKGROUND AND OBJECTIVES: We sought to determine clinical significance of neuronal septin autoimmunity and evaluate for potential IgG effects. METHODS: Septin-IgGs were detected by indirect immunofluorescence assays (IFAs; mouse tissue and cell based) or Western blot. IgG binding to (and internalization of) extracellular septin epitopes were evaluated for by live rat hippocampal neuron assay. The impact of purified patient IgGs on murine cortical neuron function was determined by recording extracellular field potentials in a multielectrode array platform. RESULTS: Septin-IgGs were identified in 23 patients. All 8 patients with septin-5-IgG detected had cerebellar ataxia, and 7 had prominent eye movement disorders. One of 2 patients with co-existing septin-7-IgG had additional psychiatric phenotype (apathy, emotional blunting, and poor insight). Fifteen patients had septin-7 autoimmunity, without septin-5-IgG detected. Disorders included encephalopathy (11; 2 patients with accompanying myelopathy, and 2 were relapsing), myelopathy (3), and episodic ataxia (1). Psychiatric symptoms (≥1 of agitation, apathy, catatonia, disorganized thinking, and paranoia) were prominent in 6 of 11 patients with encephalopathic symptoms. Eight of 10 patients with data available (from 23 total) improved after immunotherapy, and a further 2 patients improved spontaneously. Staining of plasma membranes of live hippocampal neurons produced by patient IgGs (subclasses 1 and 2) colocalized with pre- and post-synaptic markers. Decreased spiking and bursting behavior in mixed cultures of murine glutamatergic and GABAergic cortical neurons produced by patient IgGs were attributable to neither antigenic crosslinking and internalization nor complement activation. INTERPRETATION: Septin-IgGs are predictive of distinct treatment-responsive autoimmune central nervous system (CNS) disorders. Live neuron binding and induced electrophysiologic effects by patient IgGs may support septin-specific pathophysiology. ANN NEUROL 2022;92:1090-1101.

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.

Inflammatory monocytes and microglia play independent roles in inflammatory ictogenesis.

BACKGROUND: The pathogenic contribution of neuroinflammation to ictogenesis and epilepsy may provide a therapeutic target for reduction of seizure burden in patients that are currently underserved by traditional anti-seizure medications. The Theiler's murine encephalomyelitis virus (TMEV) model has provided important insights into the role of inflammation in ictogenesis, but questions remain regarding the relative contribution of microglia and inflammatory monocytes in this model. METHODS: Female C57BL/6 mice were inoculated by intracranial injection of 2 × 105, 5 × 104, 1.25 × 104, or 3.125 × 103 plaque-forming units (PFU) of the Daniel's strain of TMEV at 4-6 weeks of age. Infiltration of inflammatory monocytes, microglial activation, and cytokine production were measured at 24 h post-infection (hpi). Viral load, hippocampal injury, cognitive performance, and seizure burden were assessed at several timepoints. RESULTS: The intensity of inflammatory infiltration and the extent of hippocampal injury induced during TMEV encephalitis scaled with the amount of infectious virus in the initial inoculum. Cognitive performance was preserved in mice inoculated with 1.25 × 104 PFU TMEV relative to 2 × 105 PFU TMEV, but peak viral load at 72 hpi was equivalent between the inocula. CCL2 production in the brain was attenuated by 90% and TNFα and IL6 production was absent in mice inoculated with 1.25 × 104 PFU TMEV. Acute infiltration of inflammatory monocytes was attenuated by more than 80% in mice inoculated with 1.25 × 104 PFU TMEV relative to 2 × 105 PFU TMEV but microglial activation was equivalent between groups. Seizure burden was attenuated and the threshold to kainic acid-induced seizures was higher in mice inoculated with 1.25 × 104 PFU TMEV but low-level behavioral seizures persisted and the EEG exhibited reduced but detectable abnormalities. CONCLUSIONS: The size of the inflammatory monocyte response induced by TMEV scales with the amount of infectious virus in the initial inoculum, despite the development of equivalent peak infectious viral load. In contrast, the microglial response does not scale with the inoculum, as microglial hyper-ramification and increased Iba-1 expression were evident in mice inoculated with either 1.25 × 104 or 2 × 105 PFU TMEV. Inoculation conditions that drive inflammatory monocyte infiltration resulted in robust behavioral seizures and EEG abnormalities, but the low inoculum condition, associated with only microglial activation, drove a more subtle seizure and EEG phenotype.

Leucine Zipper 4 Autoantibody: A Novel Germ Cell Tumor and Paraneoplastic Biomarker.

OBJECTIVE: This study was undertaken to describe a novel biomarker of germ cell tumor and associated paraneoplastic neurological syndrome (PNS). METHODS: Archival sera from patients with germ cell tumor-associated PNS were evaluated. We identified a common autoantigen in a human testicular cancer cell line (TCam-2) by Western blot and mass spectrometry. Its identity was confirmed by recombinant-protein Western blot, enzyme-linked immunosorbent assay (ELISA), and cell-based assay. Autoantibody specificity was confirmed by analyzing assorted control sera/cerebrospinal fluid. RESULTS: Leucine zipper 4 (LUZP4)-immunoglobulin G (IgG) was detected in 28 patients' sera, 26 of whom (93%) were men. The median age at neurological symptom onset was 45 years (range = 28-84). Median titer (ELISA) was 1:300 (1:50 to >1:6,400, normal value < 1:50). Coexistent kelchlike protein 11-IgG was identified in 18 cases (64%). The most common presenting phenotype was rhombencephalitis (17/28, 61%). Other presentations included limbic encephalitis (n = 5, 18%), seizures and/or encephalitis (n = 2, 7%), and motor neuronopathy/polyradiculopathy (n = 4, 14%). The most common malignancy among cancer-evaluated PNS patients was seminoma (21/27, 78%). Nine of the 21 seminomas detected by whole-body fluorodeoxyglucose positron emission tomography scan (43%) were extratesticular. Both female patients had ovarian teratoma. Regressed testicular germ cell tumors were found in 4 patients. Exposure of T-cell-dendritic-cell cocultures from chronic immunosuppression-naïve LUZP4-IgG-seropositive patients to recombinant LUZP4 protein evoked a marked increase in CD69 expression on both CD4+ and CD8+ T cells when compared to vehicle-exposed and healthy control cultures. INTERPRETATION: LUZP4-IgG represents a novel serological biomarker of PNS and has high predictive value for germ cell tumors. The demonstrated antigen-specific T-cell responses support a CD8+ T-cell-mediated cytotoxic paraneoplastic and antitumor potential. ANN NEUROL 2021;89:1001-1010.

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.

Functional deficiency in endogenous interleukin-1 receptor antagonist in patients with febrile infection-related epilepsy syndrome.

OBJECTIVE: We recently reported successful treatment of a child with febrile infection-related epilepsy syndrome (FIRES), a subtype of new onset refractory status epilepticus, with the recombinant interleukin-1 (IL1) receptor antagonist (IL1RA) anakinra. On this basis, we tested whether endogenous IL1RA production or function is deficient in FIRES patients. METHODS: Levels of IL1ß and IL1RA were measured in serum and cerebrospinal fluid (CSF). The inhibitory activity of endogenous IL1RA was assessed using a cell-based reporter assay. IL1RN gene variants were identified by sequencing. Expression levels for the secreted and intracellular isoforms of IL1RA were measured in patient and control cells by real-time polymerase chain reaction. RESULTS: Levels of endogenous IL1RA and IL1ß were elevated in the serum and CSF of patients with FIRES (n = 7) relative to healthy controls (n = 10). Serum from FIRES patients drove IL1R signaling activity and potentiated IL1R signaling in response to exogenous IL1ß in a cell-based reporter assay. Functional assessment of endogenous IL1RA activity in 3 FIRES patients revealed attenuated inhibition of IL1R signaling. Sequencing of IL1RN in our index patient revealed multiple variants. This was accompanied by reduced expression of intracellular but not secreted isoforms of IL1RA in the patient's peripheral blood mononuclear cells. INTERPRETATION: Our findings suggest that FIRES is associated with reduced expression of intracellular IL1RA isoforms and a functional deficiency in IL1RA inhibitory activity. These observations may provide insight into disease pathogenesis for FIRES and other inflammatory seizure disorders and may provide a valuable biomarker for therapeutic decision-making. Ann Neurol 2019;85:526-537.

Isothermal calorimetry of a monoclonal antibody using a conventional differential scanning calorimeter.

It has been shown that isothermal calorimetry is able to provide critical information regarding the kinetics of denaturation/aggregation of monoclonal antibodies at temperatures below Tm. Those measurements, however, required sophisticated specialized instrumentation. Here, we demonstrate that similar measurements can be performed using widely available conventional differential scanning calorimeters (DSC) when operated in isothermal scan mode. The denaturation/aggregation kinetics of the anti-HIV monoclonal antibody VRC07-523LS was measured by isothermal DSC at ten degrees below Tm. It is shown that a readily available instrument provides similar kinetic information and can become an important tool for determining the long term stability of biologics.

Long term stability of a HIV-1 neutralizing monoclonal antibody using isothermal calorimetry.

Different factors affect the long term stability of monoclonal antibodies, among them denaturation or partial denaturation that is often followed by aggregation. Isothermal calorimetry is capable of quantifying the kinetics of denaturation/aggregation of an antibody by measuring the heat that is released or absorbed by the process over a period of days or weeks, at temperatures below its denaturation temperature, Tm. The denaturation/aggregation kinetics of the anti-HIV monoclonal antibody VRC07-523LS was measured by isothermal calorimetry at different concentrations in four different formulation buffers. The measurements were performed at ten degrees below Tm, as determined by differential scanning calorimetry. The formation of aggregates was also followed by size exclusion chromatography at 5 °C, 25 °C and 40 °C over a period of 8-36 weeks. It was observed that the rates measured by isothermal calorimetry correlate quantitatively with those measured by size exclusion chromatography. Since isothermal calorimetry experiments are performed over a period of ten days, it can become a valuable tool for a fast prediction of the best formulations.

Retrograde interferon-gamma signaling induces major histocompatibility class I expression in human-induced pluripotent stem cell-derived neurons.

Objective: Injury-associated axon-intrinsic signals are thought to underlie pathogenesis and progression in many neuroinflammatory and neurodegenerative diseases, including multiple sclerosis (MS). Retrograde interferon gamma (IFN γ) signals are known to induce expression of major histocompatibility class I (MHC I) genes in murine axons, thereby increasing the susceptibility of these axons to attack by antigen-specific CD8+ T cells. We sought to determine whether the same is true in human neurons. Methods: A novel microisolation chamber design was used to physically isolate and manipulate axons from human skin fibroblast-derived induced pluripotent stem cell (iPSC)-derived neuron-enriched neural aggregates. Fluorescent retrobeads were used to assess the fraction of neurons with projections to the distal chamber. Axons were treated with IFN γ for 72 h and expression of MHC class I and antigen presentation genes were evaluated by RT-PCR and immunofluorescence. Results: Human iPSC-derived neural stem cells maintained as 3D aggregate cultures in the cell body chamber of polymer microisolation chambers extended dense axonal projections into the fluidically isolated distal chamber. Treatment of these axons with IFN γ resulted in upregulation of MHC class I and antigen processing genes in the neuron cell bodies. IFN γ-induced MHC class I molecules were also anterogradely transported into the distal axon. Interpretation: These results provide conclusive evidence that human axons are competent to express MHC class I molecules, suggesting that inflammatory factors enriched in demyelinated lesions may render axons vulnerable to attack by autoreactive CD8+ T cells in patients with MS. Future work will be aimed at identifying pathogenic anti-axonal T cells in these patients.

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.

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry.

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day-1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.

CCR7 deficient inflammatory Dendritic Cells are retained in the Central Nervous System.

Dendritic cells (DC) accumulate in the CNS during neuroinflammation, yet, how these cells contribute to CNS antigen drainage is still unknown. We have previously shown that after intracerebral injection, antigen-loaded bone marrow DC migrate to deep cervical lymph nodes where they prime antigen-specific T cells and exacerbate experimental autoimmune encephalomyelitis (EAE) in mice. Here, we report that DC migration from brain parenchyma is dependent upon the chemokine receptor CCR7. During EAE, both wild type and CCR7-/- CD11c-eYFP cells infiltrated into the CNS but cells that lacked CCR7 were retained in brain and spinal cord while wild type DC migrated to cervical lymph nodes. Retention of CCR7-deficient CD11c-eYFP cells in the CNS exacerbated EAE. These data are the first to show that CD11chigh DC use CCR7 for migration out of the CNS, and in the absence of this receptor they remain in the CNS in situ and exacerbate EAE.

Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells.

Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1-CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1-IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.

Conformational stability and self-association equilibrium in biologics.

Biologics exist in equilibrium between native, partially denatured, and denatured conformational states. The population of any of these states is dictated by their Gibbs energy and can be altered by changes in physical and solution conditions. Some conformations have a tendency to self-associate and aggregate, an undesirable phenomenon in protein therapeutics. Conformational equilibrium and self-association are linked thermodynamic functions. Given that any associative reaction is concentration dependent, conformational stability studies performed at different protein concentrations can provide early clues to future aggregation problems. This analysis can be applied to the selection of protein variants or the identification of better formulation solutions. In this review, we discuss three different aggregation situations and their manifestation in the observed conformational equilibrium of a protein.

Denatured state aggregation parameters derived from concentration dependence of protein stability.

Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.