Life in the (Pluripotent) Fast Lane: Cdk1 Preserves the Epigenome to Maintain Pluripotency.

Michowski and colleagues (2020) engineered analog-sensitive Cdk1 knockin mice to identify Cdk1 targets in embryonic stem cells, which led them to discover a novel function for Cdk1 in shaping the epigenetic landscape by direct regulation of epigenetic modulators.

Persistent repair intermediates induce senescence.

Double-stranded DNA breaks activate a DNA damage checkpoint in G2 phase to trigger a cell cycle arrest, which can be reversed to allow for recovery. However, damaged G2 cells can also permanently exit the cell cycle, going into senescence or apoptosis, raising the question how an individual cell decides whether to recover or withdraw from the cell cycle. Here we find that the decision to withdraw from the cell cycle in G2 is critically dependent on the progression of DNA repair. We show that delayed processing of double strand breaks through HR-mediated repair results in high levels of resected DNA and enhanced ATR-dependent signalling, allowing p21 to rise to levels at which it drives cell cycle exit. These data imply that cells have the capacity to discriminate breaks that can be repaired from breaks that are difficult to repair at a time when repair is still ongoing.

Atopic dermatitis characteristics and medication-use patterns in school-age children with AD and asthma symptoms.

BACKGROUND: Atopic dermatitis (AD) and asthma often coexist. Both diseases can have a major impact on the lives of children with AD and their caregivers. AIM: To investigate the association of patient characteristics, comorbidities and impact of AD on children who have both asthma and AD. METHODS: Children with AD (n = 140) were selected from a larger cohort of children with a reported use of asthma medication. The Children's Dermatology Life Quality Index (CDLQI) was used to assess Quality of Life (QoL), and the Self-Assessed Eczema Area and Severity Index (SA-EASI) was used to measure AD severity. Characteristics assessed included: age, sex, and the number and type of atopic comorbidities. Medication use for AD was defined using the total number of AD prescriptions, the number of different topical AD prescriptions and the highest potency topical corticosteroid (TCS) used. Determinants of AD severity and QoL were evaluated using Spearman rank tests. RESULTS: The following factors were most strongly associated with a lower QoL: characteristics of AD lesions (Spearman Rs = 0.61-0.69, P < 0.01), a higher SA-EASI score (Rs = 0.54, P < 0.01) and a larger number of different topical AD prescriptions (Rs = 0.38, P < 0.01). The following factors were correlated with more severe AD: age (Rs = -0.36, P < 0.01), larger number of different TCS preparations used (Rs = 0.27, P < 0.05) and larger number of TCS prescriptions (Rs = 0.25, P < 0.05). CONCLUSION: In children with asthma and AD, the number of TCS preparations used is associated with lower QoL and increased AD severity.

Pharmacogenomics of inhaled corticosteroids and leukotriene modifiers: a systematic review.

BACKGROUND: Pharmacogenetics studies of anti-inflammatory medication of asthma have expanded rapidly in recent decades, but the clinical value of their findings remains limited. OBJECTIVE: To perform a systematic review of pharmacogenomics and pharmacogenetics of inhaled corticosteroids (ICS) and leukotriene modifiers (LTMs) in patients with asthma. METHODS: Articles published between 1999 and June 2015 were searched using PubMed and EMBASE. Pharmacogenomics/genetics studies of patients with asthma using ICS or LTMs were included if ≥1 of the following outcomes were studied: lung function, exacerbation rates or asthma symptoms. The studies of Single Nucleotide Polymorphisms (SNPs) that had been replicated at least once were assessed in more detail. RESULTS: In total, 59 publications were included in the systematic review: 26 addressed LTMs (including two genomewide Genome-Wide association studies [GWAS]) and 33 addressed ICS (including four GWAS). None of the GWAS reported similar results. Furthermore, none of the SNPs assessed in candidate gene studies were identified in a GWAS. No consistent reports were found for candidate gene studies of LTMs. In candidate gene studies of ICS, the most consistent results were found for rs28364072 in FCER2. This SNP was associated with all three outcomes of poor response, and the largest effect was reported with the risk of exacerbations (hazard ratio, 3.95; 95% CI, 1.64-9.51). CONCLUSION AND CLINICAL RELEVANCE: There is a lack of replication of genetic variants associated with poor ICS or LTM response. The most consistent results were found for the FCER2 gene [encoding for a low-affinity IgE receptor (CD23)] and poor ICS response. Larger studies with well-phenotyped patients are needed to assess the clinical applicability of ICS and LTM pharmacogenomics/genetics.

Genetic variation in uncontrolled childhood asthma despite ICS treatment.

Genetic variation may partly explain asthma treatment response heterogeneity. We aimed to identify common and rare genetic variants associated with asthma that was not well controlled despite inhaled corticosteroid (ICS) treatment. Data of 110 children was collected in the Children Asthma Therapy Optimal trial. Associations of genetic variation with measures of lung function (FEV1%pred), airway hyperresponsiveness (AHR) to methacholine (Mch PD20) and treatment response outcomes were analyzed using the exome chip. The 17q12-21 locus (containing ORMDL3 and GSMDB) previously associated with childhood asthma was investigated separately. Single-nucleotide polymorphisms (SNPs) in the 17q12-21 locus were found nominally associated with the outcomes. The strongest association in this region was found for rs72821893 in KRT25 with FEV1%pred (P=3.75*10(-5)), Mch PD20 (P=0.00095) and Mch PD20-based treatment outcome (P=0.006). No novel single SNPs or burden tests were significantly associated with the outcomes. The 17q12-21 region was associated with FEV1%pred and AHR, and additionally with ICS treatment response.

ST13 polymorphisms and their effect on exacerbations in steroid-treated asthmatic children and young adults.

BACKGROUND: The clinical response to inhaled corticosteroids (ICS) is associated with single nucleotide polymorphisms (SNPs) in various genes. This study aimed to relate variations in genes in the steroid pathway and asthma susceptibility genes to exacerbations in children and young adults treated with ICS. METHODS: We performed a meta-analysis of three cohort studies: Pharmacogenetics of Asthma Medication in Children: Medication with Anti-Inflammatory effects (n = 357, age: 4-12 years, the Netherlands), BREATHE (n = 820, age: 3-22 years, UK) and Paediatric Asthma Gene Environment Study (n = 391, age: 2-16 years, UK). Seventeen genes were selected based on a role in the glucocorticoid signalling pathway or a reported association with asthma. Two outcome parameters were used to reflect exacerbations: hospital visits and oral corticosteroid (OCS) use in the previous year. The most significant associations were tested in three independent validation cohorts; the Childhood Asthma Management Programme (clinical trial, n = 172, age: 5-12 years, USA), the Genes- environment and Mixture in Latino Americans II- study (n = 745, age: 8-21, USA) and the Pharmacogenetics of adrenal suppression cohort (n = 391, age: 5-18, UK) to test the robustness of the findings. Finally, all results were meta-analysed. RESULTS: Two SNPs in ST13 (rs138335 and rs138337), but not in the other genes, were associated at a nominal level with an increased risk of exacerbations in asthmatics using ICS in the three cohorts studied. In a meta-analysis of all six studies, ST13 rs138335 remained associated with an increased risk of asthma-related hospital visits and OCS use in the previous year; OR = 1.22 (P = 0.013) and OR = 1.22 (P = 0.0017), respectively. CONCLUSION AND CLINICAL RELEVANCE: A novel susceptibility gene, ST13, coding for a cochaperone of the glucocorticoid receptor, is associated with exacerbations in asthmatic children and young adults despite their ICS use. Genetic variation in the glucocorticoid signalling pathway may contribute to the interindividual variability in clinical response to ICS treatment in children and young adults.

Real world cost of human epidermal receptor 2-positive metastatic breast cancer patients: a longitudinal incidence-based observational costing study in the Netherlands and Belgium.

Currently, no country-specific metastatic breast cancer (MBC) observational costing data are available for the Netherlands and Belgium. Our aim is to describe country-specific resource use and costs of human epidermal receptor 2 (HER-2)-positive MBC in the Netherlands and Belgium, making use of real-world data. The eligibility period for patient selection was from April 2004 to April 2010. Inclusion and retrospective data collection begins at the time of first diagnosis of HER-2-positive MBC during the eligibility period and ends 24 months post-index diagnosis of MBC or at patient death. We identified 88 eligible patients in the Netherlands and 44 patients in Belgium. The total costs of medical treatment and other resource use utilisation per patient was €48,301 in the Netherlands and €37,431 in Belgium. Majority of costs was related to the use of trastuzumab in both countries, which was 50% of the total costs in the Netherlands and 56% in Belgium respectively. Our study provides estimates of resource use and costs for HER-2-positive MBC in the Netherlands and Belgium. We noticed various differences in resource use patterns between both countries demonstrating caution is needed when transferring cost estimates between countries.

Function and regulation of dynein in mitotic chromosome segregation.

Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing. How is a single motor able to fulfill this large array of functions and how are these activities temporally and spatially regulated? The answer lies in the unique composition of the dynein motor and in the interactions it makes with multiple regulatory proteins that define the time and place where dynein becomes active. Here, we will focus on the different mitotic processes that dynein is involved in, and how its regulatory proteins act to support dynein. Although dynein is highly conserved amongst eukaryotes (with the exception of plants), there is significant variability in the cellular processes that depend on dynein in different species. In this review, we concentrate on the functions of cytoplasmic dynein in mammals but will also refer to data obtained in other model organisms that have contributed to our understanding of dynein function in higher eukaryotes.

Time-dependent resource use and costs associated with different states of disease in patients diagnosed with HER-2-positive metastatic breast cancer.

Adequate reflection of disease progression and costs over time is essential in cost-effectiveness analyses based on health state-transition models. However, costing studies normally investigate the burden of metastatic breast cancer (MBC) without explicitly examining the impact of specific-disease states on health care costs over time. The objective of this study was to assess time-dependent costs of different health states of human epidermal receptor-2 (HER-2) positive MBC and the factors contributing to these costs. In the Netherlands, HER-2-positive MBC patients were identified in three different hospitals. Resource use was collected during 24 months, which was linked to unit costs and related to time with respect to date of MBC diagnosis, disease progression and death for each individual patient. Subsequently, monthly costs for different health states were calculated. Finally, a nonlinear mixed-effect modelling approach was used to provide a quantitative description of the time course of cumulative progression costs. Costs during stable disease were constant over time with a mean of $4,158. In contrast, monthly costs for progressive disease demonstrated a change over time with the largest costs in the first 2 months after diagnosis (p < 0.005). The developed mixed-effect model adequately described cumulative cost-time course and associated variability. During the last months of life, costs varied over time, with the last month of life as the most expensive one with a mean of $5,811 per patient per month. To reflect costs of HER-2-positive MBC accurately in Markov models, costs for stable disease can be defined time independent, however, costs of progressive disease should be defined time dependent, and costs related to the final months of life should be modelled as such. The mixed-effect model we have developed could now be considered for adequate description of the time-dependent cost of progressive disease.

Publication trends in newspapers and scientific journals for SSRIs and suicidality: a systematic longitudinal study.

Background In the period 2003-2008, the regulatory authorities issued several warnings restricting the use of selective serotonin re-uptake inhibitors (SSRIs) in paediatrics, in reaction to safety concerns regarding the risk of suicidality. In this study, the SSRIs and suicidality controversy serves as a template to analyse the long-term publication trends regarding the benefit/risk profile of medications. The aim is to ascertain differences (in terms of numbers, categories and timing) between negative and positive newspaper and journal articles on SSRIs and suicidality and to ascertain correlations between changes in the reports and regulatory warnings. Methods A systematic review of scientific articles (Embase) and the Netherlands (NL) and the UK newspapers (LexisNexis) was performed between 2000 and 2010. Categorisation was done by 'effect' (related treatment effect), 'type of article' and 'age group'. The articles' positive-to-negative effect ratio was determined. Differences in distribution of effect categories were analysed across sources, type of article and age group using the Mann-Whitney (two subgroups) or Kruskal-Wallis test (three or more). Findings In total, 1141 articles were categorised: 352 scientific, 224 Dutch and 565 British newspaper articles. Scientific articles were predominantly on research and were positive, whereas newspaper articles were negative (ratios=3.50-scientific, 0.69-NL and 0.94-UK; p<0.001). Articles on paediatrics were less positive in scientific journals and more negative in newspapers (ratios=2.29-scientific, 0.26-NL and 0.20-UK; p<0.001), while articles on adults were positive overall (ratios=10.0-scientific, 1.06-NL and 1.70-UK; p<0.001). In addition, negative-effect reporting trends were exacerbated following regulatory warnings and were generally opinion articles, both in scientific journals and in newspapers (2003/2004 and after 2007). Interpretation The authors found a positive publication tendency inherent in journal research articles. This apparent positive publication bias present in scientific journals, however, does not seem to prevent the dissemination of 'bad' news about medications. The negative tendency present in Dutch and British newspapers was perceivable in the paediatrics group and during the warnings, indicating that national news media have informed the public about this international drug safety controversy on time.

FCER2 T2206C variant associated with chronic symptoms and exacerbations in steroid-treated asthmatic children.

BACKGROUND: The T2206C FCER2 variant was found previously to be associated with IgE levels, exacerbation rates and decreased FCER2 expression in children on inhaled corticosteroids (ICS) participating in a clinical trial. This finding has not been replicated. We sought to replicate the association between the FCER2 gene and exacerbations in children with asthma. In addition, we tested the hypothesis that the T2206C variant may be associated with other markers of steroid resistance such as asthma symptom scores and asthma medication use. METHODS: The influence of the T2206C variant on asthma exacerbations (emergency department visits or hospitalization), symptoms scores and medication use was explored using data from two populations of asthmatic children using ICS: Pharmacogenetics of Asthma medication in Children: Medication with ANti-inflammatory effects study (n = 386) and BREATHE study (n = 939). RESULTS: The T2206C variant was associated with increased risk of asthma-related hospital visits in both cohorts (OR: 1.91, 95% CI: 1.08-3.40), and meta-analysis with previously published results was highly significant (OR: 2.38, 95% CI: 1.47-3.85, P = 0.0004). The FCER2 variant was also associated with increased risk of uncontrolled asthma measured by Asthma Control Questionnaire (OR: 2.64, 95% CI: 1.00-6.98) and was associated with increased daily steroid dose (OR: 2.46, 95% CI: 1.38-4.39). CONCLUSION: The association between the FCER2 T2206C variant and asthma-related hospitalizations in steroid-treated asthma appears robust and may also be associated with other indicators of lack of ICS efficacy such as asthma symptoms and a requirement for higher daily doses of ICS. Our results suggest that the FCER2 T2206C variant might be a useful pharmacogenetic predictor of steroid refractory patients.

Biomarkers of therapy responsiveness in asthma: pitfalls and promises.

Asthma is one of the most common chronic diseases worldwide. There is a large inter-individual variability in response to asthma treatment. Most patients respond well to standard therapy; however, a small proportion of the patients remain symptomatic despite treatment with high dosages of corticosteroids. Uncontrolled asthma leads to a decreased quality of life. Therefore, it is important to identify individuals who will respond poorly to standard asthma medication, especially to standard maintenance therapy with inhaled corticosteroids, at an early stage. Response to anti-inflammatory therapy is generally monitored by the assessment of clinical symptoms, which only partially correlates with underlying airway inflammation. The identification of specific inflammatory biomarkers might help to guide treatment or predict a corticosteroid response more accurately. Some inflammatory biomarkers are already finding their way into clinical practice (e.g. fraction of nitric oxide in exhaled breath), whereas others are predominantly used as a research tool (e.g. profiles of volatile organic compounds). Currently, there is no inflammatory biomarker used in routine clinical practice to predict a corticosteroid response. More knowledge on the underlying biological mechanism(s) of heterogeneous therapeutic responses could help to identify novel biomarkers. This review will focus on inflammatory patterns and genetic variations that may underlie differences in treatment response in patients with asthma, and will provide an overview of inflammatory biomarkers that could potentially serve as response predictors.

[Research sponsored by the industry benefits scientific progress]. / Door de industrie gesponsord onderzoek komt de wetenschappelijke vooruitgang ten goede.

The conflicts of interest dominate discussions on collaborative research between academic institutions and the pharmaceutical industry. Although one should not be blind to such possibilities and remain critical, the advantages outweigh the risks by far. The remaining diseases for which adequate pharmacotherapy is lacking are complex and require an expensive research infrastructure. Therefore, progress will benefit from collaboration and should not be hindered by short-sightedness.

Persistence with inhaled corticosteroid therapy in daily practice.

OBJECTIVE: To quantify persistence with inhaled corticosteroids (ICS) among new users in daily practice and identify determinants of persistence. METHODS: A retrospective cohort study was performed with data from the Dutch PHARMO system. This system consists of medication and hospital admission records of 325,000 inhabitants of 12 Dutch cities. In patients who were already using other drugs with a labeled indication of obstructive lung diseases (ATC: R03), individuals with a first dispensing of ICS between January 1, 1994 and December 31, 2000 were identified. Persistence with ICS was defined as the number of days on ICS treatment in the first year of use. Determinants of persistence were identified one year before start of the first dispensing of ICS. RESULTS: Approximately 50% of the patients used inhaled corticosteroids (ICS) for less than 200 days, while 18% continued treatment for one year. One-year persistence rates increased to 40% in patients with a history of multiple respiratory disease related drugs. Persistence rates also increased with lower initial doses, if the initial prescription was instituted by a medical specialist, if a patient was previously hospitalized for obstructive lung diseases, and with increasing age. CONCLUSION: The persistence rate of ICS is poor. Preventing early treatment discontinuation may be important to ensure maximal benefit from ICS treatment.

Cytokine-specific transcriptional regulation through an IL-5Ralpha interacting protein.

Cytokine receptors consist of multiple subunits, which are often shared between different receptors, resulting in the functional redundancy sometimes observed between cytokines. The interleukin 5 (IL-5) receptor consists of an IL-5-specific alpha-subunit (IL-5Ralpha) and a signal-transducing beta-subunit (betac) shared with the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. In this study, we sought to find a role for the cytoplasmic domain of IL-5Ralpha. We show that syntenin, a protein containing PSD-95/Discs large/zO-1 (PDZ) domains, associates with the cytoplasmic tail of the IL-5Ralpha. Syntenin was found to directly associate with the transcription factor Sox4. Association of syntenin with IL-5Ralpha was required for IL-5-mediated activation of Sox4. These studies identify a mechanism of transcriptional activation by cytokine-specific receptor subunits.

Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1).

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27(KIP1) through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27(KIP1) promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27(KIP1) appears to be critical in the regulation of cell survival since mere ectopic expression of p27(KIP1) was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27(KIP1) null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

STAT5-Dependent CyclinD1 and Bcl-xL expression in Bcr-Abl-transformed cells.

Signal transducers and activators of transcription (STATs) are a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. We and others have recently shown that STAT5 also plays a major role in cellular transformation by the Bcr-Abl oncogene. Here we show that the antiapoptotic bcl-xL gene product and the cell cycle regulator cyclin D1 are targets of STAT5 in Bcr-Abl-transformed cells. In the CML cell line K562 and in BaF3 cells ectopically expressing Bcr-Abl, both the cyclin D1 and bcl-x promoters are highly active. The activity of these promoters can be strongly repressed by cotransfection of a dominant negative (DN) mutant of STAT5. Moreover, the cyclin D1 and bcl-x promoters contain STAT binding sites to which STAT5 constitutively binds in Bcr-Abl transformed cells. These results suggest that STAT5 contributes to transformation by Bcr-Abl by induction of cyclin D1 and bcl-xL expression.

Identification of cytokine-regulated genes in human leukocytes in vivo.

BACKGROUND: Human polymorphic nuclear granulocytes (PMNs) such as neutrophils and eosinophils play a critical role in mediating inflammatory responses to microbial and parasitic infections. Exposure of these leukocytes to cytokines leads to an amplification of granulocyte effector functions by a mechanism termed "priming." Although many studies have investigated the effects of granulocyte priming, little is known concerning the molecular mechanisms that lead to this phenomenon. OBJECTIVE: The purpose of this study was to identify potential markers for granulocyte priming and thus also to gain further insight into the pathogenesis of inflammatory responses. METHODS: We used a modified differential display technique, random arbitrary primed-PCR to identify genes regulated during the priming of human polymorphic nuclear granulocytes by GM-CSF in vitro. Genes identified were validated by Northern blot analysis of in vitro and in vivo primed leukocytes. RESULTS: Several genes were identified and their expression characterized in vitro. One of these genes, 5-lipoxygenase-activating protein, was also found to be up-regulated in leukocytes isolated after allergen challenge of allergic asthmatic patients. CONCLUSION: The use of differential display technology is a rapid and effective means of identifying genes whose expression is regulated by priming in vitro and in vivo. Further analysis will lead to a better understanding of the priming phenotype and may provide further insight into the pathologic mechanisms of inflammatory processes.

Activation of a functionally distinct 80-kDa STAT5 isoform by IL-5 and GM-CSF in human eosinophils and neutrophils.

Interleukin-5 (IL-5), IL-3, and granulocyte macrophage colony-stimulating factor (GM-CSF) are hematopoietic cytokines which signal through a common beta subunit (betac) of a heterodimeric receptor. Among the intracellular signaling pathways activated via betac is the JAK/STAT pathway. We show that different STAT5 isoforms are activated by IL-5 and GM-CSF in eosinophils, neutrophils, and differentiated eosinophilic HL-60 cells. Whereas IL-5 activated the wild-type STAT5A and STAT5B proteins in HL60-eos cells, a carboxyl-terminally truncated 80-kDa STAT5 isoform was activated in mature eosinophils and neutrophils. Surprisingly, while both isoforms bind strongly to an element from the beta-casein promoter, only p80 STAT5 binds to the ICAM1-IRE. Consequently, a carboxyl-terminal truncated STAT5 is capable of blocking STAT3-mediated transcription of an IREtkCAT reporter construct. The cell type-specific expression of these functionally distinct STAT5 isoforms might contribute to the pleiotropic effects of IL-5 and GM-CSF on different target cells.

C/EBP regulates the promoter of the eosinophil-derived neurotoxin/RNS2 gene in human eosinophilic cells.

The eosinophil-derived neurotoxin (EDN), a member of the mammalian ribonuclease family, is found in the large specific granules of human eosinophilic leukocytes. We have investigated the role of the C/EBP transcription factor family in the regulation of EDN promoter activity. Here we show that the C/EBP family is involved in intrinsic regulation of EDN promoter activity. We have identified a C/EBP binding site located at -124 in the proximal promoter of the EDN gene. Mutation of this C/EBP site results in a decrease of promoter activity in HL-60-eos cells as well as in eosinophils differentiated in vitro from CD34+ cells. Different C/EBP proteins are able to bind to the C/EBP site as shown by gel shift assay. Our results indicate the importance of the C/EBP family in the regulation of the EDN gene in eosinophils.