In the model of Generalised Anxiety Disorder (GAD) proposed by
Dugas, Gagnon, Ladouceur, and Freeston (1998), Intolerance of Uncertainty (IU) plays a central role in the acquisition and maintenance ...of worries. A similar concept, Intolerance of Ambiguity (IA) was introduced by Frenkel-Brunswik 50
years ago. For decades, IU has been confused with IA. Researchers have applied them interchangeably. The main goal of this paper is to clarify and differentiate the notions of IU and IA, as well as to suggest new research avenues.
Most cancer cells harbor multiple drivers whose epistasis and interactions with expression context clouds drug and drug combination sensitivity prediction. We constructed a mechanistic computational ...model that is context-tailored by omics data to capture regulation of stochastic proliferation and death by pan-cancer driver pathways. Simulations and experiments explore how the coordinated dynamics of RAF/MEK/ERK and PI-3K/AKT kinase activities in response to synergistic mitogen or drug combinations control cell fate in a specific cellular context. In this MCF10A cell context, simulations suggest that synergistic ERK and AKT inhibitor-induced death is likely mediated by BIM rather than BAD, which is supported by prior experimental studies. AKT dynamics explain S-phase entry synergy between EGF and insulin, but simulations suggest that stochastic ERK, and not AKT, dynamics seem to drive cell-to-cell proliferation variability, which in simulations is predictable from pre-stimulus fluctuations in C-Raf/B-Raf levels. Simulations suggest MEK alteration negligibly influences transformation, consistent with clinical data. Tailoring the model to an alternate cell expression and mutation context, a glioma cell line, allows prediction of increased sensitivity of cell death to AKT inhibition. Our model mechanistically interprets context-specific landscapes between driver pathways and cell fates, providing a framework for designing more rational cancer combination therapy.
Objective To identify genes affected by advancing gestation and racial/ethnic origin in human ductus arteriosus (DA). Study design We collected 3 sets of DA tissue (n = 93, n = 89, n = 91; ...total = 273 fetuses) from second trimester pregnancies. We examined four genes, with DNA polymorphisms that distribute along racial lines, to identify “Caucasian” and “non-Caucasian” DA. We used real time polymerase chain reaction to measure RNA expression of 48 candidate genes involved in functional closure of the DA, and used multivariable regression analyses to examine the relationships between advancing gestation, “non-Caucasian” race, and gene expression. Results Mature gestation and non-Caucasian race are significant predictors for identifying infants who will close their patent DA when treated with indomethacin. Advancing gestation consistently altered gene expression in pathways involved with oxygen-induced constriction (eg, calcium-channels, potassium-channels, and endothelin signaling), contractile protein maturation, tissue remodeling, and prostaglandin and nitric oxide signaling in all 3 tissue sets. None of the pathways involved with oxygen-induced constriction appeared to be altered in “non-Caucasian” DA. Two genes, SLCO2A1 and NOS3 , (involved with prostaglandin reuptake/metabolism and nitric oxide production, respectively) were consistently decreased in “non-Caucasian” DA. Conclusions Prostaglandins and nitric oxide are the most important vasodilators opposing DA closure. Indomethacin inhibits prostaglandin production, but not nitric oxide production. Because decreased SLCO2A1 and NOS3 expression can lead to increased prostaglandin and decreased nitric oxide concentrations, we speculate that prostaglandin-mediated vasodilation may play a more dominant role in maintaining the “non-Caucasian” patent DA, making it more likely to close when inhibited by indomethacin.
Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide ...suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04-0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6- to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-κB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16α-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.
Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. Here, we use microwestern array to rapidly evaluate suitable conditions for ...quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use; however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Importantly, we demonstrate that results from microwestern analyses scale to normal "macro" western for a subset of antibodies.
Abstract
Background
The BRAF V600E mutation occurs in ~ twenty percent of histologically diverse pediatric gliomas and is the second most common mutation in pediatric low-grade gliomas (LGG). BRAF ...V600E expression in LGG with balanced CDKN2A is associated with a higher rate for progression than for BRAF V600E wildtype tumors, and despite adjuvant therapy, consisting of resection, radiation and chemotherapy. Progression invariably occurs in BRAF V600E mutant CDKN2A deleted gliomas, marking a high-risk group. Here, we aim to overcome the lack BRAF V600E mutant glioma models that allow for studies of stem and progenitor cells and the immune system ability to understand progression.
Methods
We develop novel immunocompetent, stem and progenitor cell-based mouse models for BRAF mutant gliomas, including genetically engineered mouse models (GEMMs), orthotopic glioma models derived from gliomas in GEMMs as well as in vitro models of those tumors. BRAF mutant mouse brains and cells were analyzed by immunofluorescence staining, flow cytometry, mass cytometry and RNA sequencing.
Results
Ongoing model development studies indicate that BRAF V600E mutant gliomas in murine brain exhibit very similar neuroanatomical preferences to human gliomas. The BRAF V600E mutation exacerbates the heterogenous cell cycling pattern of normal neural stem and progenitors and expands a symmetrically dividing progenitor population. Cellular plasticity rather than cellular lineage hierarchy drives the generation of a therapy resistant stem cell pool. Transcriptomic analyses of neuroglial stem cells with induced BRAF V600E expression provide insights into mechanisms for neoplastic transformation and progression.
Conclusion
Analyses of two independent BRAF V600E mutant mouse models provide novel insights into the role for tumor intrinsic factors, such as plasticity and stemness, and the tumor microenvironment in progression.
Abstract
Glioblastoma (GBM) is the most aggressive, invasive, and malignant primary brain cancer whose standard of care for nearly 20 years has been surgical resection followed by radiotherapy and ...temozolomide (TMZ) treatment, resulting in a median survival of 15 months following diagnosis. Heterogenous tumor cell populations that evade resection and develop chemoradiation resistance highlight the need for novel therapies that can target both the migratory and proliferative nature of GBM cells. Galectin-3, a carbohydrate-binding protein overexpressed in the GBM parenchyma and stromal astrocytes, is a potent modulator of cell proliferation, migration, angiogenesis, T-cell inhibition, and M2 macrophage polarization, and is seen to increase following TMZ and radiation treatment. Heightened Galectin-3 expression levels correlate to lower survival in glioma patients, thus greatly underscoring the need for targeting Galectin-3 in conjunction with initial standard-of-care treatment. Here, we characterize the therapeutic effects of a brain-penetrant monoclonal anti-Galectin-3 antibody (Gal3 Ab) we have developed for its ability to inhibit GBM growth and migration in several in vitro assays and in vivo heterotopic and orthotopic mouse models. Our Gal3 Ab significantly decreases cell viability, neurosphere migration, and cell invasion in vitro in the presence of full length or truncated Gal3, whose expression and secretion in GBM cells is enhanced following short and long-term TMZ stimulation. We find that Galectin-3 directly binds to EGFR, amplified in ~40% of GBM patients, as well as its most commonly mutated form, EGFRvIII, and our Gal3 Ab can block these bindings, decrease EGFR activation, and alter EGFR localization. In vivo, our Gal3 Ab decreases tumor burden and improves survival in combination with temozolomide, compared to standard-of-care treatment alone in multiple cell-line models. Our pre-clinical studies highlight the potential for our novel Gal3 antibody to further improve TMZ efficacy and benefit GBM patients by diminishing tumor cell migration and slowing tumor growth.
Abstract
Glioblastoma (GBM) is the most aggressive, invasive, and malignant primary brain cancer with a standard of care that hasn’t been improved in over 15 years. Heterogenous tumor cell ...populations that evade resection and develop resistance to chemoradiation highlight the need for novel therapies that can target both the migratory and proliferative nature of GBM cells. Galectin-3, a carbohydrate-binding protein overexpressed in the GBM parenchyma and stromal astrocytes and microglia/macrophages, is a potent modulator of cell proliferation, migration, angiogenesis, T-cell inhibition, and M2 macrophage polarization. Increased Galectin-3 expression levels correlate to lower survival in glioma patients, and these levels further increase following exposure to temozolomide or radiation alone or in combination, thus more greatly emphasizing the need to target Galectin-3 in GBM patients following initial standard-of-care treatment. Here, we characterize the therapeutic effects of a monoclonal anti-Galectin-3 antibody (Gal3 Ab) we have developed for its ability to inhibit GBM tumorigenic and metastatic potential in several in vitro assays and in vivo heterotopic and orthotopic mouse models. We find that Galectin-3 directly binds to EGFR, the most commonly amplified gene locus in GBM affecting roughly 40% of patients, and our Gal3 Ab can block this binding. Human GBM cells treated with Gal3 Ab show a significant decrease in migration and invasion and reduction in EGFR activation in vitro. In vivo, the Gal3 Ab additionally decreases tumor burden and provides a survival benefit in combination with temozolomide compared to the current standard-of-care treatment alone. Our studies indicate the potential for our novel anti-Galectin-3 antibody to further benefit GBM patients receiving chemoradiation treatment by diminishing tumor cell migration and slowing tumor growth.
Abstract
Glioblastoma (GBM) has traditionally been classified as an ‘immune cold’ tumor, characterized by strong immunosuppression, low mutational burden, and few presentable antigens. However, lack ...of standard-of-care improvement and the discovery of targetable immune-modulatory biomarkers like PD-1/PD-L1 and CTLA4 in the GBM microenvironment has prompted clinical trials to explore immune checkpoint inhibition (ICI) efficacy in patients, with moderate responses in specific patient subsets. To better characterize ICI in GBM, and to match prospective patients to suitable therapeutics, it is necessary to establish preclinical GBM mouse models that incorporate the lymphocytic milieu as xenograft and syngeneic models are unable to replicate the immune responses seen in humans. We have established a novel humanized PBMC mouse model that recapitulates human GBM pathology in the brain, supports and maintains systemic and intracranial human T cell engraftment in the presence of GBM tumors, and allows for a suitable timeline for preclinical testing of therapeutics prior to onset of GVHD. In our study, orthotopic GBM-bearing NSG mice showed successful human CD45+ engraftment in the blood (~20%) and brain (~5%) 2 weeks post intravenous implantation of hu-PBMCs. Restricted tumor growth was observed, indicating initial responsiveness of human immune cells to GBM. Engraftment remained unaffected upon treatment with temozolomide (GBM standard of care), and persisted over 6 weeks (brain ~40%, blood ~70%) before GVHD onset (week 7). ~90% of the hCD45+ population was CD3+, indicating robust T cell engraftment, which was further characterized into CD4+ and CD8+ subsets and by activation status (HLA-DR, CD45RA/RO, PD-1, FOXP3 etc) within the tumor-bearing brain and blood. Anti-PD-1 inhibitor efficacy was also tested in engrafted animals to observe ICI potential in GBM. This humanized mouse provides a powerful translational model to evaluate T cell responses in tumor pathology and immunotherapy in GBM and potentially other primary or secondary brain malignancies.
Abstract
Background
Glioblastoma (GBM) remains a largely incurable disease as current therapy fails to target the invasive nature of glioma growth in disease progression and recurrence. Here, we use ...the FDA-approved drug and small molecule Hippo inhibitor Verteporfin (VP) to target YAP-TEAD activity, known to mediate convergent aspects of tumor invasion/metastasis, and assess the drug’s efficacy and survival benefit in GBM models.
Methods
Up to 8 low-passage patient-derived GBM cell lines with distinct genomic drivers, including 3 primary/recurrent pairs, were treated with VP or vehicle (VEH) to assess in vitro effects on proliferation, migration, invasion, YAP-TEAD activity, and transcriptomics. Patient-derived orthotopic xenograft (PDX) models were used to assess VP’s brain penetrance and effects on tumor burden and survival.
Results
VP treatment disturbed YAP/TAZ-TEAD activity; disrupted transcriptome signatures related to invasion, epithelial-to-mesenchymal, and proneural-to-mesenchymal transition, phenocopying TEAD1-knockout effects; and impaired tumor migration/invasion dynamics across primary and recurrent GBM lines. In an aggressive orthotopic PDX GBM model, short-term VP treatment consistently diminished core and infiltrative tumor burden, which was associated with decreased tumor expression of Ki67, nuclear YAP, TEAD1, and TEAD-associated targets EGFR, CDH2, and ITGB1. Finally, long-term VP treatment appeared nontoxic and conferred survival benefit compared to VEH in 2 PDX models: as monotherapy in primary (de novo) GBM and in combination with Temozolomide chemoradiation in recurrent GBM, where VP treatment associated with increased MGMT methylation.
Conclusions
We demonstrate combined anti-invasive and anti-proliferative efficacy for VP with survival benefit in preclinical GBM models, indicating potential therapeutic value of this already FDA-approved drug if repurposed for GBM patients.
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