Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma.

BACKGROUND: Amplification of the MYCN oncogene is a molecular hallmark of aggressive neuroblastoma (NB), a childhood cancer of the sympathetic nervous system. There is evidence that MYCN promotes a non-inflamed and T-cell infiltration-poor ('cold') tumor microenvironment (TME) by suppressing interferon signaling. This may explain, at least in part, why patients with NB seem to have little benefit from single-agent immune checkpoint blockade (ICB) therapy. Targeting MYCN or its effectors could be a strategy to convert a cold TME into a 'hot' (inflamed) TME and improve the efficacy of ICB therapy. METHODS: NB transcriptome analyses were used to identify epigenetic drivers of a T-cell infiltration-poor TME. Biological and molecular responses of NB cells to epigenetic drugs and interferon (IFN)-γ exposure were assessed by proliferation assays, immunoblotting, ELISA, qRT-PCR, RNA-seq and ChIP-qPCR as well as co-culture assays with T cells. RESULTS: We identified H3K9 euchromatic histone-lysine methyltransferases EHMT2 and EHMT1, also known as G9a and GLP, as epigenetic effectors of the MYCN-driven malignant phenotype and repressors of IFN-γ transcriptional responses in NB cells. EHMT inhibitors enhanced IFN-γ-induced expression of the Th1-type chemokines CXCL9 and CXCL10, key factors of T-cell recruitment into the TME. In MYCN-amplified NB cells, co-inhibition of EZH2 (enhancer of zeste homologue 2), a H3K27 histone methyltransferase cooperating with EHMTs, was needed for strong transcriptional responses to IFN-γ, in line with histone mark changes at CXCL9 and CXCL10 chemokine gene loci. EHMT and EZH2 inhibitor response gene signatures from NB cells were established as surrogate measures and revealed high EHMT and EZH2 activity in MYCN-amplified high-risk NBs with a cold immune phenotype. CONCLUSION: Our results delineate a strategy for targeted epigenetic immunomodulation of high-risk NBs, whereby EHMT inhibitors alone or in combination with EZH2 inhibitors (in particular, MYCN-amplified NBs) could promote a T-cell-infiltrated TME via enhanced Th1-type chemokine expression.

Hematopoietic stem cell response to acute thrombocytopenia requires signaling through distinct receptor tyrosine kinases.

Although bone marrow niche cells are essential for hematopoietic stem cell (HSC) maintenance, their interaction in response to stress is not well defined. Here, we used a mouse model of acute thrombocytopenia to investigate the cross talk between HSCs and niche cells during restoration of the thrombocyte pool. This process required membrane-localized stem cell factor (m-SCF) in megakaryocytes, which was regulated, in turn, by vascular endothelial growth factor A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB). HSCs and multipotent progenitors type 2 (MPP2), but not MPP3/4, were subsequently activated by a dual-receptor tyrosine kinase (RTK)-dependent signaling event, m-SCF/c-Kit and VEGF-A/vascular endothelial growth factor receptor 2 (VEGFR-2), contributing to their selective and early proliferation. Our findings describe a dynamic network of signals in response to the acute loss of a single blood cell type and reveal the important role of 3 RTKs and their ligands in orchestrating the selective activation of hematopoietic stem and progenitor cells (HSPCs) in thrombocytopenia.

Golden-angle radial sparse parallel (GRASP) MRI in clinical routine detection of pituitary microadenomas: First experience and feasibility.

Background and purpose To demonstrate the clinical feasibility of a novel MRI pulse sequence, Golden-angle radial sparse parallel MRI (GRASP) through comparison to the current imaging technique, dynamic T1- weighted contrast enhanced (DCE) imaging in terms of image quality and lesion depiction in the detection of microlesions (microadenomas and cysts) of the pituitary gland. MATERIALS AND METHODS: 16 patients (11 microadenomas, 5 cysts) underwent two MRI examinations (Siemens 1.5T and 3T) on separate dates, one using standard DCE (temporal resolution 30 s) and the other using GRASP (temporal resolution of 4.4 s). Two neuroradiologists separately recorded measures of image quality (Scale 1-5, 5 = best), lesion size and contrast arrival times in terms of first and best lesion conspicuity. RESULTS: In qualitiative analysis there were no significant differences in terms of average visual image sharpness (DCE 3.9 ±â€¯0.9, GRASP 3.9 ±â€¯0.9) or visual contrast scores (DCE 4.1 ±â€¯1.2, GRASP 4.4 ±â€¯0.8). Pearson's correlation coefficients for interreader lesion measurements (width and height, mm) ranged from substantial to almost perfect agreement (r = 0.73 to 0.88). Analysis of contrast arrival times revealed an average lesion first-conspicuity time of 60.7 ±â€¯16.7 s for DCE compared to 50.2 ±â€¯10.3 s for GRASP with a difference of 10.5 ±â€¯16.2 s (p = 0.023). CONCLUSION: Depiction of pituitary microlesions is feasible with GRASP, which has the potential to increase sensitivity through higher temporal resolutions combined with isotropic acquisition allowing for multi-planar reconstructions; this remains to be proven in larger cohorts.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Structured Reporting in Neuroradiology: Intracranial Tumors.

PURPOSE: The aim of this pilot study was to assess the clinical feasibility, diagnostic yield, advantages, and disadvantages of structured reporting for routine MRI-reading in patients with primary diagnosis of intracranial tumors as compared to traditional neuroradiological free text reporting. METHODS: A structured MRI reporting template was developed covering pathological, anatomical, and functional aspects in an itemized fashion. Retrospectively, 60 consecutive patients with first diagnosis of an intracranial tumor were selected from the radiology information system/PACS system. Structured reporting was performed by a senior neuroradiologist, blinded to clinical and radiological data. Reporting times were measured per patient. The diagnostic content was compared to free text reporting which was independently performed on the same MRI exams by two other neuroradiologists. The comparisons were categorized per item as: "congruent," "partially congruent," "incongruent," or "not mentioned in free-style report." RESULTS: Tumor-related items: congruent findings were found for all items (17/17) with congruence rates ranging between 98 and 39% per item. Four items achieved congruence rates ≥90%, 5 items >80%, and 9 items ≥70%. Partially congruent findings were found for all items in up to 50% per item. Incongruent findings were present in 7/17 items in up to 5% per item. Free text reports did not mention 12 of 17 items (range 7-43% per item). Non-tumor-related items, including brain atrophy, microangiopathy, vascular pathologies, and various extracranial pathologies, which were not mentioned in free-text reports between 18 and 85% per item. Mean reporting time for structured reporting was 7:49 min (3:12-17:06 min). CONCLUSION: First results showed that expert structured reporting ensured reliable detection of all relevant brain pathologies along with reproducible documentation of all predefined diagnostic items, which was not always the case for free text reporting. A mean reporting time of 8 min per patient seems clinically feasible.

GM-CSF producing autoreactive CD4+ T cells in type 1 diabetes.

The phenotype of autoreactive T cells in type 1 diabetes is described as Th1, Th17 and/or Th21, but is largely uncharacterized. We combined multi-parameter cytokine profiling and proliferation, and identified GM-CSF producing cells as a component of the response to beta cell autoantigens proinsulin and GAD65. Overall cytokine profiles of CD4+ T cell were not altered in type 1 diabetes. In contrast, patients with recent onset type 1 diabetes had increased frequencies of proinsulin-responsive CD4+CD45RA- T cells producing GM-CSF (p=0.002), IFNγ (p=0.004), IL-17A (p=0.008), IL-21 (p=0.011), and IL-22 (p=0.007), and GAD65-responsive CD4+CD45RA- T cells producing IL-21 (p=0.039). CD4+ T cells with a GM-CSF+IFNγ-IL-17A-IL-21-IL-22- phenotype were increased in patients for responses to both proinsulin (p=0.006) and GAD65 (p=0.037). GM-CSF producing T cells are a novel phenotype in the repertoire of T helper cells in type 1 diabetes and consolidate a Th1/Th17 pro-inflammatory pathogenesis in the disease.

Targeting Adenosine in BRAF-Mutant Melanoma Reduces Tumor Growth and Metastasis.

Increasing evidence exists for the role of immunosuppressive adenosine in promoting tumor growth and spread in a number of cancer types, resulting in poor clinical outcomes. In this study, we assessed whether the CD73-adenosinergic pathway is active in melanoma patients and whether adenosine restricts the efficacy of clinically approved targeted therapies for commonly mutated BRAFV600E melanoma. In AJCC stage III melanoma patients, CD73 expression (the enzyme that generates adenosine) correlated significantly with patients presenting nodal metastatic melanoma, suggesting that targeting this pathway may be effective in advanced stage disease. In addition, dabrafenib and trametinib treatment of CD73+ BRAFV600E-mutant melanomas caused profound CD73 downregulation in tumor cells. Inhibition of BRAF and MEK in combination with the A2A adenosine receptor provided significant protection against tumor initiation and metastasis formation in mice. Our results suggest that targeting adenosine may enhance therapeutic responses for melanoma patients receiving targeted or immune-based therapies. Cancer Res; 77(17); 4684-96. ©2017 AACR.

MAPK Signaling and Inflammation Link Melanoma Phenotype Switching to Induction of CD73 during Immunotherapy.

Evolution of tumor cell phenotypes promotes heterogeneity and therapy resistance. Here we found that induction of CD73, the enzyme that generates immunosuppressive adenosine, is linked to melanoma phenotype switching. Activating MAPK mutations and growth factors drove CD73 expression, which marked both nascent and full activation of a mesenchymal-like melanoma cell state program. Proinflammatory cytokines like TNFα cooperated with MAPK signaling through the c-Jun/AP-1 transcription factor complex to activate CD73 transcription by binding to an intronic enhancer. In a mouse model of T-cell immunotherapy, CD73 was induced in relapse melanomas, which acquired a mesenchymal-like phenotype. We also detected CD73 upregulation in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade, arguing for an adaptive resistance mechanism. Our work substantiates CD73 as a target to combine with current immunotherapies, but its dynamic regulation suggests limited value of CD73 pretreatment expression as a biomarker to stratify melanoma patients. Cancer Res; 77(17); 4697-709. ©2017 AACR.

Presurgical motor, somatosensory and language fMRI: Technical feasibility and limitations in 491 patients over 13 years.

OBJECTIVES: To analyse the long-term feasibility and limitations of presurgical fMRI in a cohort of tumour and epilepsy patients with different MR-scanners at 1.5 and 3.0 T. METHODS: Four hundred and ninety-one consecutive patients undergoing presurgical fMRI between 2000 and 2012 on five different MR-scanners using established paradigms and semi-automated data processing were included. Success rates of task performance and BOLD-activation were determined for motor and somatosensory somatotopic mapping and language localisation. Procedural success, failures and imaging artifacts were analysed. MR-field strengths were compared. RESULTS: Two thousand three hundred fifteen of 2348 (98.6 %) attempted paradigms (1033 motor, 1220 speech, 95 somatosensory) were successfully performed. 100 paradigms (4.3 %) were repetition runs. 23 speech, 6 motor and 2 sensory paradigms failed for non-compliance and technical issues. Most language paradigm failures were noted in overt sentence generation. Average significant BOLD-activation was higher for motor than language paradigms (95.8 vs. 81.6 %). Most language paradigms showed significantly higher activation rates at 3 T compared to 1.5 T, whereas no significant difference was found for motor paradigms. CONCLUSIONS: fMRI proved very robust for the presurgical localisation of the different motor and somatosensory body representations, as well as Broca's and Wernicke's language areas across different MR-scanners at 1.5 and 3.0 T over 13 years. KEY POINTS: • Standardised presurgical motor and language fMRI is robust across various MRI platforms. • Motor fMRI is less dependent on field strength than language fMRI. • fMRI task failures are relatively low and are reduced by paradigm repetition.

Association between changes in cerebral grey matter volume and postoperative cognitive dysfunction in elderly patients: study protocol for a prospective observational cohort study.

BACKGROUND: Cognitive decline is frequently observed in elderly patients after major surgery. The pathophysiology of postoperative cognitive dysfunction (POCD) remains unclear. The aim of our investigation is to identify potential associations between brain volume change and POCD in elderly patients undergoing major surgery. METHODS: This is a prospective observational cohort study approved by the regional ethics board. We intend to compare specific brain volumes (hippocampus, lateral ventricle, total grey matter volume, regional cortical thickness) on magnetic resonance imaging and cognitive functions determined by a neuropsychological assessment battery in 70 study participants aged ≥65 years before and 3 and 12 months after major noncardiac surgery. Thirty volunteers will be included as matched nonsurgical controls. The primary endpoint of the study is the change in hippocampal volume over time in patients with and without POCD. The secondary endpoint is the correlation between the change in cerebral volume and cognitive function. We will follow the STROBE guidelines for reporting the results of observational studies. DISCUSSION: We hypothesize that surgery under general anesthesia is associated with a loss of cerebral grey matter, and that the degree of postoperative cognitive dysfunction correlates with the extent of atrophy in areas of the brain that are relevant for cognitive functions. The validation of reproducible anatomical biomarkers, such as the specific brain volumes examined in our cohort, may serve to evaluate the effect of preventive strategies and treatment interventions for POCD in follow-up studies. TRIAL REGISTRATION: Clinicaltrials.gov NCT02045004 . Registered 22 January 2014. Kofam.ch SNCTP000001751. Registered 21 April 2016 (retrospectively registered).

MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITF(low)/c-Jun(high) syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITF(low)/c-Jun(high) melanomas to counteract their growth-promoting and immunosuppressive functions.

Primary motor cortex activation and lateralization in patients with tumors of the central region.

Hemispheric lateralization is a frequently encountered phenomenon of cortical function. It describes the functional specialization of a region on one side of the brain for a given task. It is well characterized in motor and sensory, as well as language systems and becomes more and more known for various cognitive domains. While in the adult healthy brain hemispheric lateralization is mostly set, pathological processes may lead to cortical reorganization. In these cases neuroplasticity of the corresponding region in the non-dominant hemisphere seems to play an important role. In a previous study we investigated language associated regions in right-handed patients with frontal and temporal tumors of the left hemisphere. We observed a marked change of language lateralization in these patients towards the non-dominant hemisphere as measured by functional MRI (Partovi et al., 2012). In the present study we evaluated activation and lateralization of cortical motor areas in patients with tumors of the central region. BOLD fMRI was performed during unilateral voluntary movements of the contralesional hand in 87 patients. Individual correlations of measured BOLD-signals with the model hemodynamic reference function were determined on a ROI basis in single subjects and compared to those of 16 healthy volunteers. In volunteers the strongest activation is usually found in the M1 hand representation contralateral to the movement, while a weaker homotopic co-activation is observed in ipsilateral M1 (Stippich et al., 2007a). In the patient group our results show significant changes of motor activations, ranging from a reduction of M1 lateralization to equalization of M1 activations or even inversion of M1 lateralization during contralesional movements. This study corroborates in a large patient group the idea that lesions affecting M1 may lead to functional reorganization of cortical motor systems and in particular equalize hemispheric lateralization. However, it is not yet clear whether these changes are only an epiphenomenon or indeed reflect an attempt of recovery of brain function.

Siglec-h on activated microglia for recognition and engulfment of glioma cells.

Sialic-acid-binding immunoglobulin-like lectin-h (Siglec-h) is a recently identified mouse-specific CD33-related Siglec that signals via DAP12/TYROBP. Expression of Siglec-h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec-h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec-h by mouse microglia after interferon-γ treatment or polarization into a M1-subtype. Microglial Siglec-h acted as phagocytosis receptor since targeting of microsphere beads to Siglec-h triggered their uptake into the microglia. The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec-h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec-h and its adapter molecule DAP12. Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAP12-mediated Siglec-h dependent mechanism.

Expression of HOX genes in acute leukemia cell lines with and without MLL translocations.

In primary cells from acute leukemia patients, expression of the genes MEIS1, HOXA5, HOXA7 and HOXA9 has been reported to be correlated with the occurrence of MLL translocations. It was our aim to find out whether MLL mutant (MLLmu) and MLL wild-type (MLLwt) acute leukemia-derived cell lines might likewise be discriminated on the basis of HOX gene expression. Southern blot analysis, performed to verify the MLL status of the cells, showed that NOMO-1 was the only cell line not tested previously carrying a rearranged MLL gene. Fluorescence in situ hybridization analysis demonstrated that this cell line exhibited a reciprocal t(9;11)(q23;p22). Sequencing of RT-PCR products thereof identified unique MLL exon 10/AF-9 exon 5 fusion transcripts. We divided the acute leukemia-derived cell lines (n = 37) according to the results of Southern blot analysis into MLLmu (n = 19) and MLLwt (n = 18). Expression of HOX genes was then analyzed by applying reverse transcriptase-polymerase chain reaction, Northern and Western blot analyses. Acute myeloid leukemia (AML) cell lines expressed the HOX genes significantly more often than acute lymphoblastic (ALL) cell lines. In ALL, cells with MLL translocations expressed the genes 4 times more often than MLLwt cells. Most distinct was the correlation between MLL status and MEIS1 expression in ALL-derived cell lines: 8/8 MLLmu but 0/10 MLLwt cell lines expressed MEIS1. Northern and Western blot analysis confirmed that also HOXA9 and FLT3 were significantly more often and stronger expressed in MLLmu than in MLLwt ALL cell lines. These results suggest that MLL aberrations may regulate MEIS1 and HOXA9 gene expression in ALL-derived cell lines, while AML-derived cell lines express these genes independently of the MLL status.

Granulocyte-macrophage colony-stimulating factor: inhibitor of tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) can induce proliferation as well as apoptosis in acute myeloid leukemia (AML)-derived cells. We have shown recently that these seemingly contradictory effects are based on the divergent capacities of the cells to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) upon stimulation with TNF. Only those cells that produce GM-CSF survive the TNF attack and start growing. Here, we set out to elucidate the mechanisms of the antiapoptotic effect of GM-CSF. Protection from apoptosis was achieved by preincubating TF-1 cells with exogeneous GM-CSF. Cycloheximide prevented protection, indicating that GM-CSF might induce synthesis of antiapoptotic proteins. Regulation of protective genes was analyzed using cDNA expression arrays and the results were verified by Northern and Western blot analysis. This screen revealed the elevated expression of BCL-2, BCL-2A1, BAG-1 and TACE upon stimulation with GM-CSF. The major novelty of our study is that GM-CSF carries protective effects against TNF-induced apoptosis, not only against apoptosis induced by irradiation or cytokine-starvation. This protection requires de novo protein synthesis and is not-or at least not exclusively-the consequence of a direct crosstalk between the GM-CSF and TNF signaling pathways.