Magnus Sabel

Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production.

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57(bright) expression as a reliable phenotype of granule marker-containing CTLs. We then compared CD3(+)CD8(+)CD57(bright) CTLs with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTLs expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTLs and NK cells and suggest that analysis of CD57(bright) CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

Incidence and clinical presentation of primary hemophagocytic lymphohistiocytosis in Sweden

Primary hemophagocytic lymphohistiocytosis (HLH) represents a group of inherited hyperinflammatory immunodeficiencies, including familial HLH (FHL), Griscelli syndrome type 2 (GS2), and X‐linked lymphoproliferative syndrome (XLP). We previously reported an annual incidence of suspected primary HLH in Sweden 1971–1986 of 0.12 per 100,000 children. Here, we determined if the incidence had increased with concomitant awareness.

MethPed: a DNA methylation classifier tool for the identification of pediatric brain tumor subtypes

BackgroundClassification of pediatric tumors into biologically defined subtypes is challenging, and multifaceted approaches are needed. For this aim, we developed a diagnostic classifier based on DNA methylation profiles.ResultsMethylation data generated by the Illumina Infinium HumanMethylation 450 BeadChip arrays were downloaded from the Gene Expression Omnibus (n = 472). Using the data, we built MethPed, which is a multiclass random forest algorithm, based on DNA methylation profiles from nine subgroups of pediatric brain tumors. DNA from 18 regional samples was used to validate MethPed. MethPed was additionally applied to a set of 28 publically available tumors with the heterogeneous diagnosis PNET. MethPed could successfully separate individual histology tumor types at a very high accuracy (κ = 0.98). Analysis of a regional cohort demonstrated the clinical benefit of MethPed, as confirmation of diagnosis of tumors with clear histology but also identified possible differential diagnoses in tumors with complicated and mixed type morphology.ConclusionsWe demonstrate the utility of methylation profiling of pediatric brain tumors and offer MethPed as an easy-to-use toolbox that allows researchers and clinical diagnosticians to test single samples as well as large cohorts for subclass prediction of pediatric brain tumors. This will immediately aid clinical practice and importantly increase our molecular knowledge of these tumors for further therapeutic development.

Detection of IL-17A-producing peripheral blood monocytes in Langerhans cell histiocytosis patients

Langerhans cell histiocytosis (LCH) is a rare disease of unknown cause with manifestations ranging from isolated granulomatous lesions to life-threatening multi-system organ involvement. This disorder is further characterized by infiltration of immune cells in affected tissues and an association with interleukin (IL)-17A has been reported. Here, we investigated the presence of IL-17A-producing cells among peripheral blood mononuclear cells isolated from LCH patients and observed a high percentage of IL-17A(+) monocytes in peripheral blood of LCH patients compared to controls. The IL-17A(+) monocytes were also positive for the transcription factor retinoic acid orphan receptor (ROR) γt and showed increased mRNA levels for both IL-17A and RORγt. Notably, IL-17A was produced by all monocyte subsets and the expression level was positively associated with LCH disease activity. These data support a role for monocytes in the pathogenesis of LCH. Future therapeutic approaches may consider identification of patients who may benefit from IL-17A-targeted interventions.

A new GTF2I-BRAF fusion mediating MAPK pathway activation in pilocytic astrocytoma.

Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. A recurrent feature of PA is deregulation of the mitogen activated protein kinase (MAPK) pathway most often through KIAA1549-BRAF fusion, but also by other BRAF- or RAF1-gene fusions and point mutations (e.g. BRAFV600E). These features may serve as diagnostic and prognostic markers, and also facilitate development of targeted therapy. The aims of this study were to characterize the genetic alterations underlying the development of PA in six tumor cases, and evaluate methods for fusion oncogene detection. Using a combined analysis of RNA sequencing and copy number variation data we identified a new BRAF fusion involving the 5’ gene fusion partner GTF2I (7q11.23), not previously described in PA. The new GTF2I-BRAF 19–10 fusion was found in one case, while the other five cases harbored the frequent KIAA1549-BRAF 16–9 fusion gene. Similar to other BRAF fusions, the GTF2I-BRAF fusion retains an intact BRAF kinase domain while the inhibitory N-terminal domain is lost. Functional studies on GTF2I-BRAF showed elevated MAPK pathway activation compared to BRAFWT. Comparing fusion detection methods, we found Fluorescence in situ hybridization with BRAF break apart probe as the most sensitive method for detection of different BRAF rearrangements (GTF2I-BRAF and KIAA1549-BRAF). Our finding of a new BRAF fusion in PA further emphasis the important role of B-Raf in tumorigenesis of these tumor types. Moreover, the consistency and growing list of BRAF/RAF gene fusions suggests these rearrangements to be informative tumor markers in molecular diagnostics, which could guide future treatment strategies.

Active video gaming improves body coordination in survivors of childhood brain tumours

Abstract Purpose: We investigated whether active video gaming (AVG) could bring about regular, enjoyable, physical exercise in children treated for brain tumours, what level of physical activity could be reached and if the children’s physical functioning improved. Methods: Thirteen children, aged 7–17 years, were randomised to either AVG or waiting-list. After 10–12 weeks they crossed-over. Weekly Internet coaching sessions were used to sustain motivation and evaluate enjoyment. Energy expenditure (EE) levels were measured as Metabolic Equivalent of Task (MET), using a multisensory activity monitor. Single-blinded assessments of physical functioning were done, using the Bruininks–Osteretsky Test of Motor Performance, second edition, evaluating participants before and after the intervention period, as well as comparing the randomisation groups after the first period. Results: All patients completed the study. AVG sessions (mean duration 47 minutes) were performed on 72% of all days. Mean EE level during AVG sessions was 3.0 MET, corresponding to moderate physical activity. The Body Coordination score improved by 15% (p = 0.021) over the intervention period. Conclusions: In this group of childhood brain tumour survivors, home-based AVG, supported by a coach, was a feasible, enjoyable and moderately intense form of exercise that improved Body Coordination. Implications for Rehabilitation Childhood brain tumour survivors frequently have cognitive problems, inferior physical functioning and are less physically active compared to their healthy peers. Active video gaming (AVG), supported by Internet coaching, is a feasible home-based intervention in children treated for brain tumours, promoting enjoyable, regular physical exercise of moderate intensity. In this pilot study, AVG with Nintendo Wii improved Body Coordination.

Stem cell cultures derived from pediatric brain tumors accurately model the originating tumors

Brain tumors are the leading cause of cancer-related death in children but high-grade gliomas in children and adolescents have remained a relatively under-investigated disease despite this. A better understanding of the cellular and molecular pathogenesis of the diseases is required in order to improve the outcome for these children. In vitro-cultured primary tumor cells from patients are indispensable tools for this purpose by enabling functional analyses and development of new therapies. However, relevant well-characterized in vitro cultures from pediatric gliomas cultured under serum-free conditions have been lacking. We have therefore established patient-derived in vitro cultures and performed thorough characterization of the cells using large-scale analyses of DNA methylation, copy-number alterations and investigated their stability during prolonged time in culture. We show that the cells were stable during prolonged culture in serum-free stem cell media without apparent alterations in morphology or growth rate. The cells were proliferative, positive for stem cell markers, able to respond to differentiation cues and initiated tumors in zebrafish and mice suggesting that the cells are cancer stem cells or progenitor cells. The cells accurately mirrored the tumor they were derived from in terms of methylation pattern, copy number alterations and DNA mutations. These unique primary in vitro cultures can thus be used as a relevant and robust model system for functional studies on pediatric brain tumors.

Cell line-based xenograft mouse model of paediatric glioma stem cells mirrors the clinical course of the patient

We have established a robust and reliable cell line-based glioma stem cell xenograft mouse model, genetically and epigenetically similar to its originating patient tumours. The survival of patients and injected mice correlate significantly, demonstrating that the transplanted mice mimic the clinical course of the patient.

Correction: A new GTF2I-BRAF fusion mediating MAPK pathway activation in pilocytic astrocytoma.

[This corrects the article DOI: 10.1371/journal.pone.0175638.].

Hypothermia after cranial irradiation protects neural progenitor cells in the subventricular zone but not in the hippocampus

Abstract Purpose: To explore if hypothermia can reduce the harmful effects of ionizing radiation on the neurogenic regions of the brain in young rats. Materials and methods: Postnatal day 9 rats were randomized into two treatment groups, hypo- and normothermia, or a control group. Treatment groups were placed in chambers submerged in temperature-controlled water baths (30 °C and 36 °C) for 8 h, after receiving a single fraction of 8 Gy to the left hemisphere. Seven days’ post-irradiation, we measured the sizes of the subventricular zone (SVZ) and the granule cell layer (GCL) of the hippocampus, and counted the number of proliferating (phospho-histone H3+) cells and microglia (Iba1 + cells). Results: Irradiation caused a 53% reduction in SVZ size in the normothermia group compared to controls, as well as a reduction of proliferating cell numbers by >50%. These effects were abrogated in the hypothermia group. Irradiation reduced the number of microglia in both treatment groups, but resulted in a lower cell density of Iba1 + cells in the SVZs of the hypothermia group. In the GCL, irradiation decreased both GCL size and the proliferating cell numbers, but with no difference between the treatment groups. The number of microglia in the GCL did not change. Conclusions: Hypothermia immediately after irradiation protects the SVZ and its proliferative cell population but the GCL is not protected, one week post-irradiation.