Anne M. Cunningham

Memory function in childhood epilepsy syndromes

Objective:  Children with epilepsy are at risk of specific cognitive deficits. We aimed to compare and characterize the memory function of children with childhood absence epilepsy (CAE), frontal lobe epilepsy (FLE) and temporal lobe epilepsy (TLE).

Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease.

Objective. Various markers characterize the complex inflammatory processes seen in chronic inflammatory bowel disease (IBD) including calprotectin, a complex of two S100 proteins, which has been evaluated and validated as a faecal marker of inflammation. However, the systemic and mucosal expression patterns of calprotectin and related S100 proteins are not well characterized in this disease. The objective of this study was to assess serum and mucosal levels of calprotectin, S100A12 and soluble receptor for advanced glycation end products (sRAGE), a putative S100 ligand, in a paediatric population with IBD. Material and methods. Children were enrolled at diagnosis of IBD, along with groups of children without IBD. Standard inflammatory markers and disease activity scores were collated. Calprotectin, S100A12 and sRAGE levels in serum and biopsy culture supernatants were measured by ELISA and tissue distribution of S100 proteins was investigated by immunohistochemistry. Results. Serum and mucosal calprotectin and S100A12 levels were increased in children with IBD as compared with non-IBD controls. Serum calprotectin levels correlated with S100A12 levels and with disease activity scores in children with IBD. sRAGE levels were not increased in IBD. S100A8, S100A9 and S100A12 were abundantly expressed throughout the lamina propria and epithelium in inflamed mucosa. In contrast, these proteins were present in the lamina propria, but not the epithelium, in non-inflamed mucosa. Conclusions. Serum calprotectin and S100A12 are increased in children with IBD and indicate disease activity. Elevated levels of these proteins are present in the colonic mucosa and may contribute to the pathogenesis of IBD. Furthermore, an imbalance between sRAGE and S100A12 may contribute to inflammatory changes present in IBD.

Intelligence in childhood epilepsy syndromes

UNLABELLED Intellectual deficits play a significant role in the psychosocial comorbidity of children with epilepsy. Early educational intervention is critical. OBJECTIVE This study aims to determine the intellectual ability of children with common childhood epilepsy syndromes-generalised idiopathic epilepsy (GIE), generalised symptomatic epilepsy (GSE), temporal lobe epilepsy (TLE), frontal lobe epilepsy (FLE), central epilepsy (CE) and non-localised partial epilepsy (PE). METHODS A prospective consecutive series of 169 children were recruited. Epilepsy syndrome was identified by clinical data, seizure semiology, interictal and ictal EEG in each child, using International League Against Epilepsy criteria. Each child had neuropsychology assessment using age-normed and validated instruments. After adjusting for important epilepsy variables, 95% confidence intervals were generated for mean full-scale intelligence quotient (FSIQ) using ANCOVA. RESULTS Significant differences between epilepsy syndrome groups were found for age of onset (P<0.001), duration of active epilepsy (P=0.027), seizure frequency (P=0.037) and polytherapy (P=0.024). Analysing FSIQ, children with GIE, CE and TLE performed best, and did not differ statistically. Children with GSE had a statistically lower FSIQ than other syndrome groups except PE. FLE functioned significantly better than GSE, but did not differ statistically from other groups. CONCLUSIONS In childhood epilepsy, delineation of the syndrome has important implications when considering intellectual potential. This information is invaluable in planning educational interventions and supporting the family.

Expression of the intermediate filament protein nestin by sustentacular cells in mature olfactory neuroepithelium

The intermediate filament protein nestin has been widely used as a marker for proliferating neural progenitor cells in the nervous system. The mammalian olfactory neuroepithelium is a region of the nervous system that robustly supports ongoing neurogenesis, yet where nestin has not been reported to mark proliferating progenitors. Using immunohistochemistry, we examined nestin expression in the mature olfactory neuroepithelium and found it to be tightly restricted to the basal compartment where the olfactory neuronal progenitor cell population resides. The pattern of nestin immunoreactivity was consistent with expression by the endfeet and inferior processes of sustentacular cells rather than basal cells. Using a bank of defined antibody markers, we confirmed nestins pattern of distribution to be different from that of cytokeratin, vimentin, GBC‐1, GAP43, and carnosine. It was highly similar to the pattern of SUS‐4 immunoreactivity in the basal region of the neuroepithelium. Following surgical bulbectomy, nestin expression was up‐regulated and became evident in the cell bodies of sustentacular cells situated more apically in the neuroepithelium. We have shown nestin to be present in the basal region of the adult olfactory neuroepithelium in the zone that supports ongoing neurogenesis in the adult, but its expression is restricted to the inferior parts of sustentacular cells rather than the neuronal progenitor cells. Nestin may play a potential role in the migration of recently proliferated olfactory neurons on the scaffolding of sustentacular cells in a manner analogous to its proposed role in radial glia during embryonic development of the central nervous system. J. Comp. Neurol. 437:186–195, 2001.

TRIM16 acts as a tumour suppressor by inhibitory effects on cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein–protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β2 transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

The retinoid signalling molecule, TRIM16, is repressed during squamous cell carcinoma skin carcinogenesis in vivo and reduces skin cancer cell migration in vitro

Retinoid therapy is used for chemo‐prevention in immuno‐suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid‐sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid‐sensitive PHK cells, but not in retinoid‐resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C‐terminal RET finger protein (RFP)‐like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down‐regulated by TRIM16 and was required for TRIM16‐reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity. Copyright

Clinicopathological associations in temporal lobe epilepsy patients utilising the current ILAE focal cortical dysplasia classification

OBJECTIVES This study utilised the revised 2011 ILAE classification of focal cortical dysplasia (FCD) (Blümcke et al., 2011) to examine pathology in a cohort of children and adults who underwent temporal lobe epilepsy (TLE) surgery, and to describe the electroclinical and imaging features associated with these pathologies. METHODS The sample population were children (n=26) and adults (n=47) who underwent TLE surgery between 2002 and 2011 at our institutions. Neuropathology and MRI studies were re-reviewed by experts blinded to the original diagnosis. EEG and clinical data including current seizure outcome were determined by patient file review and/or patient contact. Pre-operative data, post-operative outcome and pathological diagnoses were compared. RESULTS The commonest pathology in the adult cohort was isolated hippocampal sclerosis (HS) (n=24, 51.1%) and in the paediatric cohort, isolated tumour (n=10, 38.5%). Overall, HS with associated FCD (FCD IIIA) was the third most common pathology (n=12, 16.4%). Temporal grey matter signal changes on MRI were associated with FCD IIIA (p=0.035). FCD IIIA had the poorest post-surgical seizure outcome compared to all other pathologies (p=0.026). A history of bilateral convulsive seizures was more common in adults (n=40, p<0.0005), and was associated with failure to achieve postoperative seizure freedom (p=0.045). Postoperatively, paediatric TLE had higher rates of seizure freedom (p=0.005) and more children had ceased medication (p<0.0005). SIGNIFICANCE FCD IIIA is a comparatively common pathological subtype in TLE, with a poor post-surgical outcome. Pre-operative recognition of FCD IIIA may be feasible through grey matter signal change on MRI. Paediatric patients had a higher rate of seizure freedom than adults. Pre-operative bilateral convulsive seizures were associated with poor outcome after surgery.

Differentiation of endogenous progenitors in an animal model of post-traumatic syringomyelia.

Study Design. An in vivo study to examine the differentiation of endogenous neural progenitor cells in an adult rat model of post-traumatic syringomyelia. Objective. To quantitatively evaluate the phenotypic fate of endogenous neural progenitor cells in post-traumatic syringomyelia. Summary of Background Data. Although neural progenitors have been identified in the central nervous system, their differentiation in experimental post-traumatic syringomyelia and possible role in the pathophysiology of this condition have not been investigated. Methods. Bromodeoxyuridine was used to label proliferating cells in a time-dependent rat model of post-traumatic syringomyelia. Eight neural markers were quantitatively analyzed to phenotype the cellular fate of these cells by double labeling immunohistochemistry. Results. Following syrinx induction, cell proliferation rate increased to 25–115 times that of cells in the intact and sham-operated controls with a peak at day 14 post-injury. In the earliest time points post-syrinx induction, ED1-expressing inflammatory cells formed a significant proportion of the proliferating population. Proliferating neural progenitor cells predominantly differentiated into NG2-expressing immature oligodendrocytes at all stages post-syrinx induction, except the final time point of 56 days. At this time, there was a peak in the number of newly generated astrocytes identified to have developed from labeled proliferating precursor cells. Conclusions. Endogenous neural progenitors proliferate markedly following induction of post-traumatic syringomyelia which consists of two stages, initial cyst formation and progressive cyst enlargement. During the former stage, macrophages proliferate in situ and contribute to the inflammatory process. The predominant cell type formed from progeny of the induced neural progenitors was characterized to be immature oliggodendrocytes. However, during the latter stage of cyst development, there was an increase in astrocytic progeny which may represent an environment more conductive to glial scar formation acting to limit further cyst enlargement.

Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 μmol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G2-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I β-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on β-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions. [Mol Cancer Ther 2008;7(9):3150–9]

Application of statistical parametric mapping to SPET in the assessment of intractable childhood epilepsy

Statistical parametric mapping (SPM) quantification and analysis has been successfully applied to functional imaging studies of partial epilepsy syndromes in adults. The present study evaluated whether localisation of the epileptogenic zone (determined by SPM) improves upon visually examined single-photon emission tomography (SPET) imaging in presurgical assessment of children with temporal lobe epilepsy (TLE) and frontal lobe epilepsy (FLE). The patient sample consisted of 24 children (15 males) aged 2.1–17.8 years (9.8±4.3 years; mean±SD) with intractable TLE or FLE. SPET imaging was acquired routinely in presurgical evaluation. All patient images were transformed into the standard stereotactic space of the adult SPM SPET template prior to SPM statistical analysis. Individual patient images were contrasted with an adult control group of 22 healthy adult females. Resultant statistical parametric maps were rendered over the SPM canonical magnetic resonance imaging (MRI). Two corresponding sets of ictal and interictal SPM and SPET images were then generated for each patient. Experienced clinicians independently reviewed the image sets, blinded to clinical details. Concordance of the reports between SPM and SPET images, syndrome classification and MRI abnormality was studied. A fair level of inter-rater reliability (kappa=0.73) was evident for SPM localisation. SPM was concordant with SPET in 71% of all patients, the majority of the discordance being from the FLE group. SPM and SPET localisation were concordant with epilepsy syndrome in 80% of the TLE cases. Concordant localisation to syndrome was worse for both SPM (33%) and SPET (44%) in the FLE group. Data from a small sample of patients with varied focal structural pathologies suggested that SPM performed poorly relative to SPET in these cases. Concordance of SPM and SPET with syndrome was lower in patients younger than 6 years than in those aged 6 years and above. SPM is effective in localising the potential epileptogenic zone but does not provide additional benefit beyond SPET in presurgical assessment of children with intractable epilepsy. The impact of different pathologies on the efficacy of SPM warrants further study.