Human CD4+CD25+ T cells expressing a chimeric antigen receptor against aberrant superoxide dismutase 1 trigger antigen-specific immunomodulation.

BACKGROUND AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs' immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. METHODS: A CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. RESULTS: The CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. CONCLUSIONS: Taken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

Exploiting natural killer group 2D receptors for CAR T-cell therapy.

Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

Early inflammatory mediator gene expression in two models of traumatic brain injury: ex vivo cortical slice in mice and in vivo cortical impact in piglets.

BACKGROUND: The immunological response during the first 24 hours after traumatic brain injury (TBI) may be a critical therapeutic interval for limiting the secondary neuronal damage that is influenced by enhanced inflammatory mediator expression. METHODS: To gain further insight of the early injury response, we examined the expression of several inflammatory genes by real-time qPCR as a function of time or distance from injury in two distinct mammalian models: an ex vivo mouse cortical slice injury system and an in vivo piglet model of brain injury. RESULTS: Interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), chemokine ligands 2 (CCL2), 3 (CCL3), 4 (CCL4), and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNAs increased within 5 h after injury in mouse cortical slices. Chemokine and PTGS2 mRNAs remained elevated in slices at 24 h, whereas IL-1ß and TNF-α expressions decreased from earlier peak levels. At 24 h after cortical injury in 1-month-old piglets, the expression of CCL2 mRNA was significantly increased in the lesion core and in the penumbra region. The expression of PTGS2, IL-1ß, and TNF-α was variable among the piglets. CONCLUSIONS: These in vitro and large animal models of cortical injury expand our understanding of the early timing and spread of the immunological response and can serve as preclinical systems to facilitate the discovery of therapeutic agents for TBI aimed at regulating inflammatory mediator expression.

A novel mouse model of Niemann-Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations.

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.

Neurodegeneration by activated microglia across a nanofiltration membrane.

Microglia have been implicated in the pathogenesis of several neurodegenerative diseases, but their precise role remains elusive. Although neuron loss in the presence of lipopolysaccharide-stimulated microglia has been well documented, a novel coculture paradigm was developed as a new approach to assess the diffusible, soluble mediators of neurodegeneration. Isolated microglia were plated on membrane inserts that were coated with a layer of cellulose acetate. The cellulose acetate-coated membranes have nanofiltration properties, in that only molecules with masses less than 350 Da can pass through. Products released from activated microglia that were separated from primary ventral mesencephalon cells beneath the nanofiltering membrane were able to kill the dopamine neurons. Microglial cytokines cannot diffuse through this separating membrane. Addition of a nitric oxide synthase inhibitor prevented the loss of the dopamine neurons. These data describe a novel coculture system for studying diffusible factors and further support nitric oxide production as an important mediator in microglia-induced neuron death.

Managing emergency department patients with recent-onset atrial fibrillation.

BACKGROUND: The management of emergency department (ED) patients with presumed recent-onset atrial fibrillation or flutter≤48h in duration varies widely. OBJECTIVE AND METHOD: We conducted a prospective study across three affiliated community EDs within a large integrated health care delivery system to describe the management of patients with recent-onset atrial fibrillation or flutter, to determine the safety and effectiveness of ED cardioversion, and to measure the incidence of thromboembolism 30 days after discharge. RESULTS: We enrolled 206 patients with convenience sampling between June 2005 and November 2007. Mean age was 64.0±14.4 years (range 21-96 years). Patients were grouped for analysis into four categories based on whether cardioversion was 1) spontaneous in the ED (59; 28.6%); 2) attempted with electrical or pharmacological means (115; 56.3%), with success in 110 (95.7%); 3) hoped for during a short stint of home observation (16; 7.8%, 11 of which spontaneously converted to sinus rhythm within 24h); or 4) contraindicated (16; 7.8%). Of the entire group, 183 (88.8%) patients were discharged home. Adverse events requiring ED interventions were reported in 6 (2.9%; 95% confidence interval [CI] 1.1-6.2%) patients, all of whom recovered. Two (1.0%; 95% CI 0.1-3.5%) patients were found to have an embolic event on 30-day follow-up. CONCLUSIONS: Our approach to ED patients with presumed recent-onset atrial fibrillation or flutter seems to be safe and effective, with a high rate of cardioversion and discharge to home coupled with a low ED adverse event and 30-day thromboembolic event rate.

Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system.

BACKGROUND: While it is clear that inbred strains of mice have variations in immunological responsiveness, the influence of genetic background following tissue damage in the central nervous system is not fully understood. A cortical explant system was employed as a model for injury to determine whether the immediate transcriptional response to tissue resection revealed differences among three mouse strains. METHODS: Immunological mRNAs were measured in cerebral cortex from SJL/J, C57BL/6J, and BALB/cJ mice using real time RT-PCR. Freshly isolated cortical tissue and cortical sections incubated in explant medium were examined. Levels of mRNA, normalized to ß-actin, were compared using one way analysis of variance with pooled samples from each mouse strain. RESULTS: In freshly isolated cerebral cortex, transcript levels of many pro-inflammatory mediators were not significantly different among the strains or too low for comparison. Constitutive, baseline amounts of CD74 and antisecretory factor (ASF) mRNAs, however, were higher in SJL/J and C57BL/6J, respectively. When sections of cortical tissue were incubated in explant medium, increased message for a number of pro-inflammatory cytokines and chemokines occurred within five hours. Message for chemokines, IL-1α, and COX-2 transcripts were higher in C57BL/6J cortical explants relative to SJL/J and BALB/cJ. IL-1ß, IL-12/23 p40, and TNF-α were lower in BALB/cJ explants relative to SJL/J and C57BL/6J. Similar to observations in freshly isolated cortex, CD74 mRNA remained higher in SJL/J explants. The ASF mRNA in SJL/J explants, however, was now lower than levels in both C57BL/6J and BALB/cJ explants. CONCLUSIONS: The short-term cortical explant model employed in this study provides a basic approach to evaluate an early transcriptional response to neurological damage, and can identify expression differences in genes that are influenced by genetic background.

Progressive changes in microglia and macrophages in spinal cord and peripheral nerve in the transgenic rat model of amyotrophic lateral sclerosis.

BACKGROUND: The role of neuroinflammation in motor neuron death of amyotrophic lateral sclerosis (ALS) is unclear. The human mutant superoxide dismutase-1 (hmSOD1)-expressing murine transgenic model of ALS has provided some insight into changes in microglia activity during disease progression. The purpose of this study was to gain further knowledge by characterizing the immunological changes during disease progression in the spinal cord and peripheral nerve using the more recently developed hmSOD1 rat transgenic model of ALS. METHODS: Using immunohistochemistry, the extent and intensity of tissue CD11b expression in spinal cord, lumbar nerve roots, and sciatic nerve were evaluated in hmSOD1 rats that were pre-clinical, at clinical onset, and near disease end-stage. Changes in CD11b expression were compared to the detection of MHC class II and CD68 microglial activation markers in the ventral horn of the spinal cord, as well as to the changes in astrocytic GFAP expression. RESULTS: Our study reveals an accumulation of microglia/macrophages both in the spinal cord and peripheral nerve prior to clinical onset based on CD11b tissue expression. The microglia formed focal aggregates in the ventral horn and became more widespread as the disease progressed. Hypertrophic astrocytes were not prominent in the ventral horn until after clinical onset, and the enhancement of GFAP did not have a strong correlation to increased CD11b expression. Detection of MHC class II and CD68 expression was found in the ventral horn only after clinical onset. The macrophages in the ventral nerve root and sciatic nerve of hmSOD1 rats were observed encircling axons. CONCLUSIONS: These findings describe for the first time in the hmSOD1 rat transgenic model of ALS that enhancement of microglia/macrophage activity occurs pre-clinically both in the peripheral nerve and in the spinal cord. CD11b expression is shown to be a superior indicator for early immunological changes compared to other microglia activation markers and astrogliosis. Furthermore, we suggest that the early activity of microglia/macrophages is involved in the early phase of motor neuron degeneration and propose that studies involving immunomodulation in hmSOD1transgenic models need to consider effects on macrophages in peripheral nerves as well as to microglia in the spinal cord.

Efficacy of thalidomide for the treatment of amyotrophic lateral sclerosis: a phase II open label clinical trial.

Neuroinflammation through the cytokine, tumor necrosis factor-alpha (TNF-alpha) is thought to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). We conducted a preliminary phase II trial of thalidomide, which reduces levels of TNF-alpha pre-transcriptionally and post-transcriptionally in vivo and has been shown to prolong disease duration and extend the lifespan of transgenic animal models of ALS. Patients who met diagnostic criteria for ALS received thalidomide at escalating doses to a target dose of 400 mg/day. The primary endpoints in the trial were the ALS Functional Rating Scale (ALSFRS) and pulmonary function testing (PFT) curves after nine months of thalidomide treatment that were compared to historical controls. Secondary endpoints were: survival stratified for newly diagnosed and progressive disease, toxicity, quality of life, and serum cytokine measurements. Twenty-three patients were enrolled, but only 18 were evaluable for the primary outcome. There was no improvement in the ALSFRS or PFT compared to historical controls. Thalidomide had several side-effects in our ALS patients. There was no significant shift in cytokine profile after treatment compared to baseline. In conclusion, treatment of ALS with the TNF-alpha inhibitor, thalidomide, does not appear to effectively modulate disease progression and can cause adverse effects.

Neuromyelitis optica pathogenesis and aquaporin 4.

Neuromyelitis optica (NMO) is a severe, debilitating human disease that predominantly features immunopathology in the optic nerves and the spinal cord. An IgG1 autoantibody (NMO-IgG) that binds aquaporin 4 (AQP4) has been identified in the sera of a significant number of NMO patients, as well as in patients with two related neurologic conditions, bilateral optic neuritis (ON), and longitudinal extensive transverse myelitis (LETM), that are generally considered to lie within the NMO spectrum of diseases. NMO-IgG is not the only autoantibody found in NMO patient sera, but the correlation of pathology in central nervous system (CNS) with tissues that normally express high levels of AQP4 suggests NMO-IgG might be pathogenic. If this is the case, it is important to identify and understand the mechanism(s) whereby an immune response is induced against AQP4. This review focuses on open questions about the "events" that need to be understood to determine if AQP4 and NMO-IgG are involved in the pathogenesis of NMO. These questions include: 1) How might AQP4-specific T and B cells be primed by either CNS AQP4 or peripheral pools of AQP4? 2) Do the different AQP4-expressing tissues and perhaps the membrane structural organization of AQP4 influence NMO-IgG binding efficacy and thus pathogenesis? 3) Does prior infection, genetic predisposition, or underlying immune dysregulation contribute to a confluence of events which lead to NMO in select individuals? A small animal model of NMO is essential to demonstrate whether AQP4 is indeed the incipient autoantigen capable of inducing NMO-IgG formation and NMO. If the NMO model is consistent with the human disease, it can be used to examine how changes in AQP4 expression and blood-brain barrier (BBB) integrity, both of which can be regulated by CNS inflammation, contribute to inductive events for anti-AQP4-specific immune response. In this review, we identify reagents and experimental questions that need to be developed and addressed to enhance our understanding of the pathogenesis of NMO. Finally, dysregulation of tolerance associated with autoimmune disease appears to have a role in NMO. Animal models would allow manipulation of hormone levels, B cell growth factors, and other elements known to increase the penetrance of autoimmune disease. Thus an AQP4 animal model would provide a means to manipulate events which are now associated with NMO and thus demonstrate what set of events or multiplicity of events can push the anti-AQP4 response to be pathogenic.

Advances in the use of natural receptor- or ligand-based chimeric antigen receptors (CARs) in haematologic malignancies.

Chimeric antigen receptors (CAR)-T cell therapy has recently made promising advances towards treatment of B-cell malignancies. This approach makes use of an antibody-derived single chain variable fragment (scFv)-based CAR to target the CD19 antigen. Currently scFvs are the most common strategy for creation of CARs, but tumor cells can also be targeted using non-antibody based approaches with designs focused on the interaction between natural receptors and their ligands. This emerging strategy has been used in unique ways to target multiple tumor types, including solid and haematological malignancies. In this review, we will highlight the performance of receptor-ligand combinations as designs for CARs to treat cancer, with a particular focus on haematologic malignancies.

Regulatory T cell-based therapies for autoimmunity.

Autoimmune disorders are long-term diseases that adversely affect the quality of life for patients, and they are one of the top ten leading causes of death. While each autoimmune disorder is unique, they all are caused by a breakdown of tolerance against endogenous proteins. This leads to auto-inflammatory events that promote the destruction of organs in a humoral and cellular immune mediated manner. Treatment options for autoimmunity can involve the use of chemical and biologic agents that suppress inflammation. While these treatment options for patients have shown to be beneficial in autoimmunity, they can result in patients being vulnerable to opportunistic infections. Newer therapies aim to identify methods to specifically block auto-inflammatory immune cells while allowing for an intact immune response to other antigens. T regulatory (Treg) cells are a subtype of the adoptive immune cell that is capable of suppressing inflammatory events in an antigen-specific manner, but they are often poorly functioning within autoimmune patients. Treg cells have been well characterized for their immune modulating capabilities and preclinical and early clinical studies support their therapeutic potential for antigen-specific immune suppression. This review will examine the current understanding of Treg cell function and the therapeutic potential of enhancing Treg cells in patients with inflammatory disorders.

Purification and culture of spinal motor neurons.

Motor neurons are responsible for voluntary movement. Lower motor neurons are characterized by large soma, the potential to form very long axons, and wide-ranging dendritic arborization. They receive direction from various neuronal cell types and induce movement of skeletal muscle fibers through acetylcholine release at the neuromuscular junction. Each lower motor neuron can communicate with 10 to several hundred muscle fibers at firing rates modulated by the balance of ongoing neurotransmitter signaling. Disease and trauma that affect lower motor neurons can cause paralysis and, in some cases, death. Studies using primary cultures of these cells have ongoing potential to facilitate a deeper understanding of their biology and function.

Purification and culture of spinal motor neurons from rat embryos.

We describe an immunopanning protocol to isolate, enrich, and culture spinal motor neurons from rat embryonic spinal cords. The method takes advantage of several distinct properties of rat lower motor neurons to isolate them from neighboring cells. First, an ideal stage in development after motor neurons are born (embryonic day 14 during rat gestation), but prior to extensive axonal extension or developmental apoptosis, is exploited. Lower motor neurons cannot be viably isolated using this method after birth. After dissociating embryonic spinal cord tissue, which contains lower motor neurons among many other cell types, the uniquely large motor neurons are enriched using density gradient centrifugation. Finally, the collected cell population is further purified based on selective immunopanning for motor neurons, which express the low-affinity nerve growth factor (NGF) receptor often referred to as p75. The near-pure lower motor neuron cultures are plated and seeded in defined conditions optimal for survival and can be maintained for several weeks. The expected yield is approximately 70,000 cells per embryonic spinal cord.

Anti-inflammatory efficacy of dexamethasone and Nrf2 activators in the CNS using brain slices as a model of acute injury.

Limiting excessive production of inflammatory mediators is an effective therapeutic strategy for many diseases. It's also a promising remedy for neurodegenerative diseases and central nervous system (CNS) injuries. Glucocorticoids are valuable anti-inflammatory agents, but their use is constrained by adverse side-effects. Activators of NF-E2-related factor-2 (Nrf2) signaling represent an attractive anti-inflammatory alternative. In this study, dexamethasone, a synthetic glucocorticoid, and several molecular activators of Nrf2 were evaluated for efficacy in slices of cerebral cortex derived from adult SJL/J mice. Cortical explants increased expression of IL-1ß and TNF-α mRNAs in culture within 5 h of sectioning. This expression was inhibited with dexamethasone in the explant medium or injected systemically in mice before sectioning. Semi-synthetic triterpenoid (SST) derivatives, potent activators of the Nrf2 pathway, demonstrated fast-acting anti-inflammatory activity in microglia cultures, but not in the cortical slice system. Quercetin, luteolin, and dimethyl fumarate were also evaluated as molecular activators of Nrf2. While expression of inflammatory mediators in microglia cultures was inhibited, these compounds did not demonstrate anti-inflammatory efficacy in cortical slices. In conclusion, brain slices were amenable to pharmacological modification as demonstrated by anti-inflammatory activity with dexamethasone. The utilization of Nrf2 activators to limit inflammatory mediators within the CNS requires further investigation. Inactivity in CNS tissue, however, suggests their safe use without neurological side-effects in treating non-CNS disorders. Short-term CNS explants may provide a more accurate model of in vivo conditions than microglia cultures since the complex tissue microenvironment is maintained.

Synthetic triterpenoid CDDO derivatives modulate cytoprotective or immunological properties in astrocytes, neurons, and microglia.

2-Cyano-3,12-dioxoolean-1, 9-dien-28-oic acid (CDDO) is a semisynthetic triterpenoid. CDDO derivatives with an amide, butyl ester (BE), imidazolide (IM), or trifluoroethyl amide (TFEA) group at position C-28 of CDDO were evaluated in glial and neuronal cells, in vitro. Changes in intracellular NADPH:quinone oxidoreductase (NQO1) levels, protection against oxidative toxicity, endotoxin-induced free-radical production, and the median lethal concentration (LC50) were assessed. All four CDDO derivatives at nanomolar concentrations increased NQO1 levels in astrocytes and moderately in neurons, but not in microglial cells. Pretreatment with 100 nM of CDDO-amide, CDDO-TFEA, or CDDO-IM protected astrocytes from hydrogen peroxide toxicity. Only CDDO-amide protected neuronal cells. Pretreatment of microglial cells with CDDO derivatives at nanomolar concentrations attenuated endotoxin-induced nitric oxide protection. The effectiveness for NQO1 induction, protection against oxidative toxicity, and attenuation of nitric oxide production, as well as cell viability at higher concentrations, varied among the derivatives with different functional groups at C-28. CDDO-amide had comparable or even a greater effectiveness at altering cytoprotective and immunomodulatory properties while having higher LC50 values for each neural cell type examined. These results indicate that derivatives of CDDO modulate important pathways relevant to many neurological diseases that involve both chronic inflammation and free-radical damage with variable effects based on the functional group at C-28 and cell type.

Afferent influences on striatal development in organotypic cocultures.

Organotypic cocultures of striatum, cortex, and ventral mesencephalon were used to study the anatomical and physiological development of striatal neurons in the presence or absence of cortical and nigral (SN/VTA) inputs. Striatum and cortex were dissected from prenatal (E18-E22) or early postnatal (P0-P2) rats, and SN/VTA was dissected from E14-15 fetuses; pieces were maintained up to 3 weeks in static slice culture. Triple cocultures containing SN/VTA exhibited rapid and robust dopamine (DA) innervation of the striatum in a patchy pattern, and homogeneous distribution within the cortical piece, regardless of the orientations of the three pieces. DA fibers within the striatal piece overlapped striatal patch neurons, marked by DARPP-32 immunoreactivity, in striatal cultures prepared from all age rats, but development most analogous to that seen in vivo was observed with the use of late prenatal (E20-E22) striatum. The patch/matrix organization was maintained in cultures prepared from late prenatal striatum in the presence of cortical and nigrostriatal DA afferents. In addition, a more complete transition to a patchy organization was observed in E18/19 striatal cultures in the presence of cortical and DA innervation. Electrophysiological recording demonstrated the presence of both spontaneous and cortically evoked activity in striatal medium spiny neurons; this activity was greatly influenced by the presence of DA innervation. These findings demonstrate the importance of afferent innervation in the maturation of striatal neurons in organotypic cultures.

A telephone advice line does not decrease the number of presentations to Christchurch Emergency Department, but does decrease the number of phone callers seeking advice.

AIM: To describe the effect of a pilot national telephone advice service (Healthline) on a public hospital emergency department. METHODS: We prospectively gathered information from the Christchurch Hospital Emergency Department (ED) computer- and non-computer-based information systems, for a six-month period during the operation of Healthline. We compared the data with five earlier periods when Healthline was not running. In addition, Healthline collected and analysed data from call log information. RESULTS: There was a small increase (1.1%) in ED attendance during the study period. Patients referred by Healthline had a similar triage distribution to the general ED population, but a lower admission rate (29% vs 47%). Telephone calls to the ED dropped dramatically during the study period. CONCLUSIONS: Healthline had little effect on overall ED census and appeared to refer patients with similar acuity to the general ED census. It decreased the workload for ED nursing staff charged with answering advice calls.