Serum IgG-induced microglial activation enhances neuronal cytolysis via the NO/sGC/PKG pathway in children with opsoclonus-myoclonus syndrome and neuroblastoma.

BACKGROUND: Opsoclonus-myoclonus syndrome (OMS) is a rare neurological disease. Some children with OMS also have neuroblastoma (NB). We and others have previously documented that serum IgG from children with OMS and NB induces neuronal cytolysis and activates several signaling pathways. However, the mechanisms underlying OMS remain unclear. Here, we investigated whether nitric oxide (NO) from activated microglias and its cascade contribute to neuronal cytolysis in pediatric OMS. METHODS: The activation of cultured cerebral cortical and cerebellar microglias incubated with sera or IgG isolated from sera of children with OMS and NB was measured by the expression of the activation marker, cytokines, and NO. Neuronal cytolysis was determined after exposing to IgG-treated microglia-conditioned media. Using inhibitors and activators, the effects of NO synthesis and its intracellular cascade, namely soluble guanylyl cyclase (sGC) and protein kinase G (PKG), on neuronal cytolysis were evaluated. RESULTS: Incubation with sera or IgG from children with OMS and NB increased the activation of cerebral cortical and cerebellar microglias, but not the activation of astrocytes or the cytolysis of glial cells. Moreover, the cytolysis of neurons was elevated by conditioned media from microglias incubated with IgG from children with OMS and NB. Furthermore, the expression of NO, sGC, and PKG was increased. Neuronal cytolysis was relieved by the inhibitors of NO signaling, while neuronal cytolysis was exacerbated by the activators of NO signaling but not proinflammatory cytokines. The cytolysis of neurons was suppressed by pretreatment with the microglial inhibitor minocycline, a clinically tested drug. Finally, increased microglial activation did not depend on the Fab fragment of serum IgG. CONCLUSIONS: Serum IgG from children with OMS and NB potentiates microglial activation, which induces neuronal cytolysis through the NO/sGC/PKG pathway, suggesting an applicability of microglial inhibitor as a therapeutic candidate.

IGF-1 alleviates serum IgG-induced neuronal cytolysis through PI3K signaling in children with opsoclonus-myoclonus syndrome and neuroblastoma.

BACKGROUND: Opsoclonus-myoclonus syndrome (OMS) is a rare neurological disorder, usually accompanied by neuroblastoma (NB). There is no targeted treatment and animal model of OMS. We aimed to investigate whether insulin-like growth factor 1 (IGF-1)/phosphoinositide 3-kinase (PI3K) signaling alleviates neuronal cytolysis in pediatric OMS. METHODS: Cultured rat cerebral cortical neurons and cerebellar neurons were incubated with sera or IgG isolated from sera of children with OMS and NB. Cytolysis and PI3K expression were measured by the lactate dehydrogenase assay and enzyme-linked immunosorbent assay, respectively. Using inhibitors and activators, the effects of IGF-1 and PI3K on cytolysis were investigated. RESULTS: The incubation of sera or IgG from children with OMS and NB increased cytolysis in not only cerebellar neurons, but also cerebral cortical neurons. Furthermore, the IGF-1 receptor antagonist NVP-AEW541 exaggerated cytolysis in children with OMS and NB. IGF-1 alleviated cytolysis, which was blocked by the PI3K inhibitor LY294002. Additionally, sera or IgG from children with OMS and NB compensatively elevated PI3K expression. LY294002 exacerbated cytolysis; whereas, the PI3K activator 740 Y-P suppressed cytolysis. CONCLUSION: IGF-1/PI3K signaling alleviates the cytolysis of cultured neurons induced by serum IgG from children with OMS and NB, which may be innovation therapy targets.

Autoantibody-mediated cytotoxicity in paediatric opsoclonus-myoclonus syndrome is dependent on ERK-1/2 phophorylation.

Paediatric opsoclonus-myoclonus syndrome (OMS) is in 50% of the cases associated with a neuroblastoma as a paraneoplastic syndrome and is associated with surface-binding antibodies against cerebellar granular neurons (CGN). To evaluate possible pathogenic effects of these autoantibodies on CGN we examined their influence on the MAPKinase enzymes ERK-1/2 and p38 using flow cytometry and phospho-specific antibodies. OMS IgG but not IgG from neuroblastoma without OMS or healthy controls induced phosphorylation of ERK-1/2 in cerebellar granular neurons (p<0.01). No effect on p38 phosphorylation or on HEK293 control cell line could be detected. IgG-mediated phosphorylation of ERK-1/2 was associated with an increased cytotoxicity of CGN, which could be blocked by ERK-1/2 pathway inhibitor U0126. We here show that IgG-mediated anti-neuronal cytotoxicity in OMS is mediated by ERK-1/2 phosphorylation in CGN.

CCR7 signaling in pediatric opsoclonus-myoclonus: upregulated serum CCL21 expression is steroid-responsive.

Identifying and blocking chemokine inflammatory mediators in pediatric opsoclonus-myoclonus syndrome (OMS) is critical to the treatment of this autoimmune, paraneoplastic, neurological disorder. In a prospective, case-control, clinico-scientific study of children with OMS compared to non-inflammatory neurological controls and other inflammatory neurological disorders, CCL19 (n=369) and CCL21 (n=312) were quantified in CSF and serum, respectively, by ELISA. Both cross-sectional and longitudinal effects of OMS and various immunotherapies were evaluated. Significant upregulation of CCL21 concentration (mean ± SD) (+32%) was found in serum of untreated OMS (630 ± 133 pg/mL), compared to controls (478 ± 168 pg/mL), (p<0.0001). Both corticosteroids and ACTH (corticotropin) significantly lowered CCL21 to control levels, as they did in combination with IVIg, rituximab, cyclophosphamide or other treatments, without additional reduction attributable to the other agents. In a pilot longitudinal study of ACTH-based triple therapy, the mean serum CCL21 concentration fell 59% from elevated to less than 1 SD below controls 1 week after high-dose ACTH, gradually returning to the control mean with ACTH tapering by 3 weeks and out to 12 weeks (p<0.0001). In contrast, CCL19, detectable in CSF, was not significantly altered by OMS or various immunotherapies. In the "high" CCL21 subgroup, higher serum concentrations of CCL22 (+57%) and CXCL13 (+40%), as well as the CSF concentration of BAFF (+64%), also were found. Elevated serum CCL21, not CSF CCL19, correlates with OMS severity and duration in pediatric OMS. Corticosteroids and ACTH were the only immunotherapies evaluated that down-regulated CCL21 production. Validation studies are needed to assess treatment biomarker status.

Role of BAFF in Opsoclonus-Myoclonus syndrome, a bridge between cancer and autoimmunity.

OMS is a rare paraneoplastic disorder that affects adults and children. Pediatric OMS is often associated with NB, a common, solid tumor of childhood, derived from the sympathetic nervous system. The detection of autoantibodies and lymphocytic infiltration in NB patients led to advance an autoimmune hypothesis for the pathogenesis of OMS-related NB. BAFF is a potent modulator of B cell growth and survival upon interaction with its receptors BAFF-R and BCMA. The aim of this study was to investigate mechanism(s) involved in ectopic lymphoid neogenesis in OMS-associated NB. We investigated BAFF, BAFF-R, and BCMA expression in NB tumors associated or not with OMS. Furthermore, we evaluated BAFF expression and secretion in NB cell lines, treated or untreated with differentiating agents. Immunohistochemically, lymphocytes infiltrating NB tumors from patients, with or without OMS, expressed BAFF, BAFF-R, and BCMA, whereas neuroblasts expressed BAFF and BCMA but not BAFF-R. By flow cytometry, BAFF was found to be consistently expressed in NB cell lines. Similarly to the results obtained in tissue lesions, BCMA but not BAFF-R was detected on the surface of all NB cell lines under basal conditions. De novo synthesis of BAFF-R and up-regulation of BCMA were observed in NB cell lines upon treatment with IFN-γ or 13-cis retinoic acid. This study provides new insights in the mechanisms driving the neogenesis of lymphoid follicles and in the functional interactions between tumor and immune cells in OMS-associated NB.

Protein-RNA and protein-protein recognition by dual KH1/2 domains of the neuronal splicing factor Nova-1.

Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We report the crystal structure of the first two KH domains (KH1/2) of Nova-1 bound to an in vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site. Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. Whereas the canonical RNA-binding surface of KH2 in the above complex engages in protein-protein interactions in the crystalline state, the individual KH2 domain can sequence-specifically target the UCAC RNA element in solution. The observed antiparallel alignment of KH1 and KH2 domains in the crystal structure of the complex generates a scaffold that could facilitate target pre-mRNA looping on Nova binding, thereby potentially explaining Nova's functional role in splicing regulation.

Pediatric dosing of rituximab revisited: serum concentrations in opsoclonus-myoclonus syndrome.

To longitudinally assess serum concentrations of rituximab, it was administered intravenously to 25 children with opsoclonus-myoclonus syndrome at 375 mg/m2 on each of 4 consecutive weeks with (Group I and II) or without (Group III) conventional immunotherapy. Serum rituximab levels, drawn before and after each infusion and at later intervals, were analyzed by enzyme-linked immunosorbent assay. Rituximab concentration increased stepwise with each infusion, dropping by the next infusion, thereby forming 4 discrete peaks (Cmax) and troughs (Cmin). It then fell precipitously to trace levels at 4 months. However, Cmax and Cmin curves differed significantly between groups. Compared with the youngest children (Group I), the oldest (Group III) had a 34% lower rituximab concentration at the fourth infusion, 45% less IgM depletion 1 month later, and received 20% less rituximab when the dose was recalculated as mg/kg. Serum IgM and rituximab levels were negatively correlated. Peak rituximab concentration did not correlate with adrenocorticotropic hormone dose. These results indicate that the degree of serum IgM depletion is a useful indicator for rituximab dose equivalency in children of different ages. They also suggest that pediatric rituximab dosing should be based on body weight, not surface area. (ClinicalTrials.gov NCT00244361).

Clonazepam responsive opsoclonus myoclonus syndrome: additional evidence in favour of fastigial nucleus disinhibition hypothesis?

Opsoclonus myoclonus syndrome is a rare paraneoplastic syndrome seen in 50% of children with neuroblastoma. Neural generator of opsoclonus and myoclonus is not known but evidences suggest the role of fastigial nucleus disinhibition from the loss of function of inhibitory (GABAergic) Purkinje cells in the cerebellum. We present a child with paraneoplastic opsoclonus myoclonus syndrome who responded well to clonazepam. Response to clonazepam is an evidence for the involvement of GABAergic neural circuits in the genesis of opsoclonus myoclonus syndrome and is in agreement with fastigial nucleus disinhibition hypothesis.

Neurometabolic effects of ACTH on free amino compounds in opsoclonus-myoclonus syndrome.

To evaluate the possible role of central free amino compounds in pediatric opsoclonus-myoclonus syndrome (OMS), 21 cerebrospinal fluid (CSF) amino compounds were measured by an amino acid analyzer or mass spectroscopy in 74 anesthetized children, 54 with OMS and 20 age-matched neurological controls. In OMS, only phosphoethanolamine was increased compared to controls; OMS severity and duration had significant converse effects on alanine and phosphoethanolamine. In contrast, corticotropin (ACTH) treatment was associated with increased alanine and phenylalanine, and decreased taurine compared to controls and untreated OMS, and increased glutamine, lysine, ornithine, and tyrosine compared to untreated OMS. Other than low taurine, these effects were not found with corticosteroid treatment, and non-steroidogenic immunotherapy had no effect. The ACTH dose-association was most apparent for alanine and phosphoethanolamine, but lysine and ornithine were also higher in the high-dose ACTH group. There were no significant disease- or treatment-associated perturbations in GABA, glycine, or other amino acids. These data suggest a unique pattern of ACTH effects on non-neurotransmitter CSF amino compounds, for the most part not shared by steroids.