Quantitative Models of the Mechanisms That Control Genome-Wide Patterns of Transcription Factor Binding during Early Drosophila Development

Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision

Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34+ CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34+ CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34+ CP-CML cell engraftment in NSG mice and, upon administration to EGFP+ /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells

In vitro testing to diagnose venom allergy and monitor immunotherapy: a placebo-controlled, crossover trial

9600 GARSINGTON RD, OXFORD, ENGLAND, OXON, OX4 2D

A meta‐analysis on allergen‐specific immunotherapy using MCT ®

BACKGROUND: The World Allergy Organization and the European Academy of Allergy and Clinical Immunology recommend to perform product‐specific meta‐analyses for allergen‐specific immunotherapies because of the high degree of heterogeneity between individual products. This meta‐analysis evaluates the efficacy and safety of Glutaraldehyde‐modified and MCT(®) (MicroCrystalline Tyrosine)‐adsorbed allergoids (MATA). METHODS: The databases MEDLINE, LILACS, embase, LIVIVO, Web of Science and Google (Scholar) were searched for publications on MATA up to June 2019. Primary endpoint was the combined symptom and medication score (CSMS). Secondary endpoints were single scores, immunogenicity and improvement of allergic condition. Secondary safety endpoints were the occurrence of side effects. A random effects model was applied with (standardized) mean differences ([S]MDs) including confidence intervals (CI). Heterogeneity was analyzed using the I(2) index and publication bias using Egger's test and Funnel plots. Subgroups were analyzed regarding age and asthma status. RESULTS: Eight randomized double‐blind placebo‐controlled trials were selected for efficacy and 43 publications for safety analysis. In total, 4531 patients were included in this analysis including eight studies containing data on children and adolescents. AIT with MATA significantly reduced allergic symptoms and medication use with a SMD for CSMS of −0.8 (CI: −1.24, −0.36) in comparison to placebo. Heterogeneity was moderate between the studies. The total symptom score (−1.2 [CI: −2.11, −0.29]) and the total medication score (−2.2 [CI: −3.65, −0.74]) were also significantly reduced after MATA treatment. Patient's condition improved significantly after treatment with MATA, with an odds ratio of 3.05 (CI: 1.90, 4.90) when compared to placebo. The proportion of patients, who developed side effects was 38% (CI: 19%, 57%). No serious side effects occurred. Safety in the subgroups of asthmatic patients, children and adolescents did not differ from the overall patient population. CONCLUSIONS: This meta‐analysis reveals a large body of evidence from publications investigating MATA. MATA significantly improved allergic symptoms and reduced the use of anti‐allergic medication in comparison to placebo, with an excellent safety profile. Especially for children and asthmatic patients, the use of MATAs can be considered as safe, because the safety profiles in these groups did not differ from the total patient population

Endothelial cell and cAMP regulation of T-cell CD40 ligand: relevance of calcium/calmodulin-dependent kinase IV signalling

CD40 ligand (CD40L) expression is now recognized to contribute in the pathogenesis of vascular disease. Because increased CD40L has been associated with myocardial infarction, effects of endothelial cells and cAMP with respect to CD40L regulation may be of clinical relevance. In the present study, endothelial cells are shown to markedly increase CD40L on naïve CD4(+) T cells with a more modest effect on memory T cells. Furthermore, the addition of dibutyryl cyclic AMP (dbcAMP) synergistically increased naïve cell CD40L but inhibited memory cell CD40L. Although it has previously been recognized that human endothelial cells can increase T-cell CD40L, this is the first description of the difference in responses of naïve and memory cells and the first demonstration of synergistic effects of endothelial cells and cAMP on CD40L regulation. Consistent with previous reports that CD40L regulation is distinctive, another marker of early activation (CD69) was not similarly regulated. The mechanisms of CD40L regulation were related to calcineurin and calcium/calmodulin dependent kinase IV (CaMKIV) signalling pathways. Endothelial cell costimulation of CD40L was found to be dependent upon calcineurin activity while cAMP actions to increase CD40L were dependent upon CaMKIV. Expression of a dominant negative CaMKIV construct further indicated an important role for CaMKIV in regulation of CD40L and cAMP responses. These data indicate that endothelial cell costimulation can interact with cAMP through calcium signalling pathways to synergistically enhance CD40L expression. Because increased CD40L is associated with atherosclerotic plaque and instability, results are relevant to the pathogenesis of atherosclerosis and myocardial infarction

Irradiation up-regulates CD80 expression through induction of tumour necrosis factor-α and CD40 ligand expression on B lymphoma cells

Previously, we reported that 100 Gy X-ray irradiation followed by 24 hr incubation up-regulates CD80 expression in murine B lymphoma cells, A20-2J. In the present study, we analysed the underlying mechanisms of such up-regulation using A20-HL cells derived from A20-2J cells. Irradiation of A20-HL cells with 100 Gy enhanced CD80 expression. Incubation of untreated A20-HL cells with those 100 Gy irradiated induced up-regulation of CD80 expression. Irradiation of A20-HL cells also up-regulated the expression of tumour necrosis factor-α (TNF-α) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF-α and CD40L in cell lysates. The addition of anti-TNF-α or anti-CD40L monoclonal antibody (mAb) to the incubation of irradiated A20-HL cells partially inhibited up-regulation of CD80 expression, and the addition of both antibodies together almost completely inhibited the up-regulation, suggesting that irradiation up-regulated the CD80 expression through the induction of TNF-α and CD40L expression. Irradiation also increased the accumulation of CD80, TNF-α and CD40L mRNA. n-tosyl-l-phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)-κB inhibitor, markedly decreased irradiation-induced accumulation of CD80 mRNA and CD80 expression. FK506, a calcineurin inhibitor, and nifedipine, a calcium channel inhibitor, inhibited not only the expression of TNF-α and CD40L, but also the up-regulation of CD80 on irradiated A20-HL cells. These results strongly suggested that irradiation induced TNF-α and CD40L expression, which then up-regulated CD80 mRNA and CD80 expression through activation of NF-κB transcription factor in A20-HL cells