Autochthonous Case of Pulmonary Histoplasmosis, Switzerland.

In Europe, pulmonary histoplasmosis is rarely diagnosed except in travelers. We report a probable autochthonous case of severe chronic pulmonary histoplasmosis in an immunocompetent man in Switzerland without travel history outside of Europe. Diagnosis was achieved by histopathology, fungal culture, and serology, but the source of the infection remains speculative

The CXCR4/CXCR7/CXCL12 Axis Is Involved in a Secondary but Complex Control of Neuroblastoma Metastatic Cell Homing.

Neuroblastoma (NB) is one of the most deadly solid tumors of the young child, for which new efficient and targeted therapies are strongly needed. The CXCR4/CXCR7/CXCL12 chemokine axis has been involved in the progression and organ-specific dissemination of various cancers. In NB, CXCR4 expression was shown to be associated to highly aggressive undifferentiated tumors, while CXCR7 expression was detected in more differentiated and mature neuroblastic tumors. As investigated in vivo, using an orthotopic model of tumor cell implantation of chemokine receptor-overexpressing NB cells (IGR-NB8), the CXCR4/CXCR7/CXCL12 axis was shown to regulate NB primary and secondary growth, although without any apparent influence on organ selective metastasis. In the present study, we addressed the selective role of CXCR4 and CXCR7 receptors in the homing phase of metastatic dissemination using an intravenous model of tumor cell implantation. Tail vein injection into NOD-scid-gamma mice of transduced IGR-NB8 cells overexpressing CXCR4, CXCR7, or both receptors revealed that all transduced cell variants preferentially invaded the adrenal gland and typical NB metastatic target organs, such as the liver and the bone marrow. However, CXCR4 expression favored NB cell dissemination to the liver and the lungs, while CXCR7 was able to strongly promote NB cell homing to the adrenal gland and the liver. Finally, coexpression of CXCR4 and CXCR7 receptors significantly and selectively increased NB dissemination toward the bone marrow. In conclusion, CXCR4 and CXCR7 receptors may be involved in a complex and organ-dependent control of NB growth and selective homing, making these receptors and their inhibitors potential new therapeutic targets

Low number of neurosecretory vesicles in neuroblastoma impairs massive catecholamine release and prevents hypertension.

Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system. It produces and releases metanephrines, which are used as biomarkers for diagnosis in plasma and urine. However, plasma catecholamine concentrations remain generally normal in children with NB. Thus, unlike pheochromocytoma and paraganglioma (PHEO/PGL), two other non-epithelial neuroendocrine tumors, hypertension is not part of the usual clinical picture of patients with NB. This suggests that the mode of production and secretion of catecholamines and metanephrines in NB is different from that in PHEO/PGL, but little is known about these discrepancies. Here we aim to provide a detailed comparison of the biosynthesis, metabolism and storage of catecholamines and metanephrines between patients with NB and PHEO. Catecholamines and metanephrines were quantified in NB and PHEO/PGL patients from plasma and tumor tissues by ultra-high pressure liquid chromatography tandem mass spectrometry. Electron microscopy was used to quantify neurosecretory vesicles within cells derived from PHEO tumor biopsies, NB-PDX and NB cell lines. Chromaffin markers were detected by qPCR, IHC and/or immunoblotting. Plasma levels of metanephrines were comparable between NB and PHEO patients, while catecholamines were 3.5-fold lower in NB vs PHEO affected individuals. However, we observed that intratumoral concentrations of metanephrines and catecholamines measured in NB were several orders of magnitude lower than in PHEO. Cellular and molecular analyses revealed that NB cell lines, primary cells dissociated from human tumor biopsies as well as cells from patient-derived xenograft tumors (NB-PDX) stored a very low amount of intracellular catecholamines, and contained only rare neurosecretory vesicles relative to PHEO cells. In addition, primary NB expressed reduced levels of numerous chromaffin markers, as compared to PHEO/PGL, except catechol O-methyltransferase and monoamine oxidase A. Furthermore, functional assays through induction of chromaffin differentiation of the IMR32 NB cell line with Bt2cAMP led to an increase of neurosecretory vesicles able to secrete catecholamines after KCl or nicotine stimulation. The low amount of neurosecretory vesicles in NB cytoplasm prevents catecholamine storage and lead to their rapid transformation by catechol O-methyltransferase into metanephrines that diffuse in blood. Hence, in contrast to PHEO/PGL, catecholamines are not secreted massively in the blood, which explains why systemic hypertension is not observed in most patients with NB

TWIST1 expression is associated with high-risk neuroblastoma and promotes primary and metastatic tumor growth.

The embryonic transcription factors TWIST1/2 are frequently overexpressed in cancer, acting as multifunctional oncogenes. Here we investigate their role in neuroblastoma (NB), a heterogeneous childhood malignancy ranging from spontaneous regression to dismal outcomes despite multimodal therapy. We first reveal the association of TWIST1 expression with poor survival and metastasis in primary NB, while TWIST2 correlates with good prognosis. Secondly, suppression of TWIST1 by CRISPR/Cas9 results in a reduction of tumor growth and metastasis colonization in immunocompromised mice. Moreover, TWIST1 knockout tumors display a less aggressive cellular morphology and a reduced disruption of the extracellular matrix (ECM) reticulin network. Additionally, we identify a TWIST1-mediated transcriptional program associated with dismal outcome in NB and involved in the control of pathways mainly linked to the signaling, migration, adhesion, the organization of the ECM, and the tumor cells versus tumor stroma crosstalk. Taken together, our findings confirm TWIST1 as promising therapeutic target in NB

Rocking Promotes Sleep in Mice through Rhythmic Stimulation of the Vestibular System.

Rocking has long been known to promote sleep in infants and, more recently, also in adults, increasing NREM sleep stage N2 and enhancing EEG slow waves and spindles. Nevertheless, whether rocking also promotes sleep in other species, and what the underlying mechanisms are, has yet to be explored. In the current study, C57BL/6J mice equipped with EEG and EMG electrodes were rocked laterally during their main sleep period, i.e., the 12-h light phase. We observed that rocking affected sleep in mice with a faster optimal rate than in humans (1.0 versus 0.25 Hz). Specifically, rocking mice at 1.0 Hz increased time spent in NREM sleep through the shortening of wake episodes and accelerated sleep onset. Although rocking did not increase EEG activity in the slow-wave and spindle-frequency ranges in mice, EEG theta activity (6-10 Hz) during active wakefulness shifted toward slower frequencies. To test the hypothesis that the rocking effects are mediated through the vestibular system, we used the otoconia-deficient tilted (tlt) mouse, which cannot encode linear acceleration. Mice homozygous for the tlt mutation were insensitive to rocking at 1.0 Hz, while the sleep and EEG response of their heterozygous and wild-type littermates resembled those of C57BL/6J mice. Our findings demonstrate that rocking also promotes sleep in the mouse and that this effect requires input from functional otolithic organs of the vestibule. Our observations also demonstrate that the maximum linear acceleration applied, and not the rocking rate per se, is key in mediating the effects of rocking on sleep

Novel HTS Strategy Identifies TRAIL-Sensitizing Compounds Acting Specifically Through the Caspase-8 Apoptotic Axis

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is potentially a very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to its cognate receptors, Death Receptors-4 and -5, leads to recruitment of caspase-8 and classical activation of downstream effector caspases, leading to apoptosis. As with many drugs however, TRAIL's usefulness is limited by resistance, either innate or acquired. We describe here the development of a novel 384-well high-throughput screening (HTS) strategy for identifying potential TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner. By utilizing a TRAIL resistant cell line lacking caspase-8 (NB7) compared to the same cells reconstituted with the wild-type protein, or with a catalytically inactive point mutant of caspase-8, we are able to identify compounds that act specifically through the caspase-8 axis, rather than through general toxicity. In addition, false positive hits can easily be “weeded out” in this assay due to their activity in cells lacking caspase-8-inducible activity. Screening of the library of pharmacologically active compounds (LOPAC) was performed as both proof-of-concept and to discover potential unknown TRAIL sensitizers whose mechanism is caspase-8 mediated. We identified known TRAIL sensitizers from the library and identified new compounds that appear to sensitize specifically through caspase-8. In sum, we demonstrate proof-of-concept and discovery of novel compounds with a screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers. This screen was performed in the 384-well format, but could easily be further miniaturized, allows easy identification of artifactual false positives, and is highly scalable to accommodate diverse libraries

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1).

In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations