Low-dose theophylline enhances the anti-inflammatory effects of steroids during exacerbations of chronic obstructive pulmonary disease

ABSTRACT Background: Chronic obstructive pulmonary disease (COPD) is characterised by an abnormal inflammatory response mainly to cigarette smoke that flares up during exacerbations of the disease (ECOPD). Reduced activity of histone deacetylases (HDAC) contributes to enhanced inflammation in stable COPD. It was hypothesised that HDAC activity is further reduced during ECOPD and that theophylline, an HDAC activator, potentiates the antiinflammatory effect of steroids in these patients. A study was performed to investigate HDAC activity during ECOPD and the effects of theophylline on the anti-inflammatory effects of steroids in a randomised single-blind controlled study. Methods: 35 patients hospitalised with ECOPD and treated according to international guidelines (including systemic steroids) were randomised to receive or not to receive low-dose oral theophylline (100 mg twice daily). Before treatment and 3 months after discharge, HDAC and nuclear factor-kB (NF-kB) activity in sputum macrophages, the concentration of nitric oxide in exhaled air (eNO) and total antioxidant status (TAS), tumour necrosis factor a (TNFa), interleukin (IL)-6 and IL8 levels in sputum supernatants were measured. Results: Patients receiving standard therapy showed decreased NF-kB activity, eNO concentration and sputum levels of TNFa, IL6 and IL8, as well as increased TAS during recovery of ECOPD, but HDAC activity did not change. The addition of low-dose theophylline increased HDAC activity and further reduced IL8 and TNFa concentrations. Conclusions: During ECOPD, low-dose theophylline increases HDAC activity and improves the anti-inflammatory effects of steroids.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

The function of a new regulator of epidermal differentiation, HOP, and pathomechanisms underlying epidermolytic hyperkeratosis

The process of epidermal differentiation involves proliferation, differentiation, migration and maturation of keratinocytes to form an impermeable barrier against water loss and outside environment. It is controlled by highly balanced regulatory machinery, involving many molecules that are still under investigation.Homeobox proteins are involved in body patterning and morphogenesis of organs and are studied as potentially good candidates to regulate this process. In the first project we investigated the role of a protein named HOP which belongs to a group of homeobox proteins. Even if HOP is a small protein almost completely composed of the homeodomain and without DNA binding capacity, it is considered as transcriptional regulator in different tissues. HOP interacts with serum response factor (SRF) and histone deacetylase type 2 (HDAC2). By microarray analysis we found that HOP expression increases in cultured human primary keratinocytes (NHK) which undergo calcium-induced differentiation. HOP protein was localized in granular layer of the epidermis of healthy individuals. Lack of HOP was demonstrated in psoriatic lesions, whereas a strong expression was demonstrated in the lesional skin of patients affected with lichen planus (LP). Since LP is characterized by hypergranulosis while psoriatic lesions by progressive lack of the granular layer, the obtained data indicated that HOP might have a potential function in granular layer of epidermis. To investigate HOP function, we inhibited its expression by using HOP specific StealthRNAi and we overexpressed HOP using lentiviral vectors in differentiating NHK. The conclusion of both experiments indicated that HOP positively regulates the expression of late differentiation markers, such as profilaggrin, loricrin and transglutaminase 1. The in vitro data were next confirmed in vivo using HOP knockout mouse model.The second part of my study involved analysis of mechanisms underlying the pathogenesis of epidermolytic hyperkeratosis (EHK). EHK is a genetic disorder characterized by erythema, skin blistering, keratinocyte hyperproliferation and hyperkeratosis. EHK is caused by mutations in keratin 1 or 10 (K1, K10) which are major structural proteins of differentiated keratinocytes and participate in the cellular scaffold formation. To investigate how the structural proteins carrying mutations alter cellular signaling, we established an in vitro model for EHK by overexpression of one of the most common K10 mutations reported so far (K10R156H), in primary human keratinocytes. In order to mimic the in vivo situation, mutated keratinocytes growing on silicone membranes were subjected to mechanical stretch. We observed strong collapse of KIF in K10R156H keratinocytes when subjected to stretch for 30 minutes. Our data demonstrated stronger activation of p38, a member of MAPK stress signaling pathways, in K10R156H when compared to control cells. We demonstrated also that K10R156H keratinocytes showed an induction of TNF-α and RANTES release in response to stretch.Taken together these studies characterize a novel regulator of epidermal differentiation - HOP and demonstrate new aspects implicated in the pathogenesis of EHK.

A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes.

The Nrf2 transcription factor controls the expression of genes involved in the antioxidant defense system. Here, we identified Nrf2 as a novel regulator of desmosomes in the epidermis through the regulation of microRNAs. On Nrf2 activation, expression of miR-29a and miR-29b increases in cultured human keratinocytes and in mouse epidermis. Chromatin immunoprecipitation identified the Mir29ab1 and Mir29b2c genes as direct Nrf2 targets in keratinocytes. While binding of Nrf2 to the Mir29ab1 gene activates expression of miR-29a and -b, the Mir29b2c gene is silenced by DNA methylation. We identified desmocollin-2 (Dsc2) as a major target of Nrf2-induced miR-29s. This is functionally important, since Nrf2 activation in keratinocytes of transgenic mice causes structural alterations of epidermal desmosomes. Furthermore, the overexpression of miR-29a/b or knockdown of Dsc2 impairs the formation of hyper-adhesive desmosomes in keratinocytes, whereas Dsc2 overexpression has the opposite effect. These results demonstrate that a novel Nrf2-miR-29-Dsc2 axis controls desmosome function and cutaneous homeostasis.

No evidence for mutations of CTCFL/BORIS in Silver-Russell syndrome patients with IGF2/H19 imprinting control region 1 hypomethylation.

BACKGROUND: Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease. METHODOLOGY/PRINCIPAL FINDINGS: DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing. CONCLUSIONS/SIGNIFICANCE: As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.

EORTC topics in neurooncology: The long path from a focus on neurological complications of cancer towards molecularly defined trials and therapies in neurooncology

Over the past decade a series of trials of the EORTC Brain Tumor Group (BTG) has substantially influenced and shaped the standard-of-care of primary brain tumors. All these trials were coupled with biological research that has allowed for better understanding of the biology of these tumors. In glioblastoma, EORTC trial 26981/22981 conducted jointly with the National Cancer Institute of Canada Clinical Trials Group showed superiority of concomitant radiochemotherapy with temozolomide over radiotherapy alone. It also identified the first predictive marker for benefit from alkylating agent chemotherapy in glioblastoma, the methylation of the O6-methyl-guanyl-methly-transferase (MGMT) gene promoter. In another large randomized trial, EORTC 26951, adjuvant chemotherapy in anaplastic oligodendroglial tumors was investigated. Despite an improvement in progression-free survival this did not translate into a survival benefit. The third example of a landmark trial is the EORTC 22845 trial. This trial led by the EORTC Radiation Oncology Group forms the basis for an expectative approach to patients with low-grade glioma, as early radiotherapy indeed prolongs time to tumor progression but with no benefit in overall survival. This trial is the key reference in deciding at what time in their disease adult patients with low-grade glioma should be irradiated. Future initiatives will continue to focus on the conduct of controlled trials, rational academic drug development as well as systematic evaluation of tumor tissue including biomarker development for personalized therapy. Important lessons learned in neurooncology are to dare to ask real questions rather than merely rapidly testing new compounds, and the value of well designed trials, including the presence of controls, central pathology review, strict radiology protocols and biobanking. Structurally, the EORTC BTG has evolved into a multidisciplinary group with strong transatlantic alliances. It has contributed to the maturation of neurooncology within the oncological sciences.

Expression of O⁶-methylguanine-DNA methyltransferase in childhood medulloblastoma.

Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O(6)-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In seven MB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7-92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely (n = 46; Pearson correlation r (2) = 0.14, P = 0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT'91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should not.

P16 Mediated Suppression Of Telomerase In Normal And Malignant Human Breast Cells.

Summary The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal.

Personalized care in neuro-oncology coming of age: why we need MGMT and 1p/19q testing for malignant glioma patients in clinical practice.

Histological subtyping and grading by malignancy are the cornerstones of the World Health Organization (WHO) classification of tumors of the central nervous system. They shall provide clinicians with guidance as to the course of disease to be expected and the choices of treatment to be made. Nonetheless, patients with histologically identical tumors may have very different outcomes, notably in patients with astrocytic and oligodendroglial gliomas of WHO grades II and III. In gliomas of adulthood, 3 molecular markers have undergone extensive studies in recent years: 1p/19q chromosomal codeletion, O(6)-methylguanine methyltransferase (MGMT) promoter methylation, and mutations of isocitrate dehydrogenase (IDH) 1 and 2. However, the assessment of these molecular markers has so far not been implemented in clinical routine because of the lack of therapeutic implications. In fact, these markers were considered to be prognostic irrespective of whether patients were receiving radiotherapy (RT), chemotherapy, or both (1p/19q, IDH1/2), or of limited value because testing is too complex and no chemotherapy alternative to temozolomide was available (MGMT). In 2012, this situation has changed: long-term follow-up of the Radiation Therapy Oncology Group 9402 and European Organisation for Research and Treatment of Cancer 26951 trials demonstrated an overall survival benefit from the addition to RT of chemotherapy with procarbazine/CCNU/vincristine confined to patients with anaplastic oligodendroglial tumors with (vs without) 1p/19q codeletion. Furthermore, in elderly glioblastoma patients, the NOA-08 and the Nordic trial of RT alone versus temozolomide alone demonstrated a profound impact of MGMT promoter methylation on outcome by therapy and thus established MGMT as a predictive biomarker in this patient population. These recent results call for the routine implementation of 1p/19q and MGMT testing at least in subpopulations of malignant glioma patients and represent an encouraging step toward the development of personalized therapeutic approaches in neuro-oncology.

Mitotic figure counts are overrated in resection specimen of invasive breast carcinoma

In order for some patients to benefit from aggressive chemotherapy for invasive breast carcinoma, many patients are currently being treated without little or no benefit. Enormous effort is hence being directed towards the identification of those patients who will need chemotherapy and those who will not. Since chemotherapy targets proliferating cells pathologists focus on the proliferative activity of tumors, as assessed by mitotic figure counts or by cell cycle specific immunohistochemical markers, such as Ki-67 and H3 histone. As far as the tumor grade is concerned, many of these studies have reported a tendency to up-grade carcinomas in resection specimen when compared to the initial diagnosis on the biopsy material, and most studies have noted that the upgrade in resection specimen is due solely or to a large extent to an increase in the mitotic figure count. In the present study, we propose a different explanation for the divergence in mitotic figure counts between biopsy and resection material. We assessed the proliferative activity of 52 invasive ductal carcinomas and confirm that the number of mitotic figures significantly increased by a factor of more than 3 in resection specimen over the biopsy material, while at the same time the pan-cell cycle specific marker MIB-1 yieldes comparable results. we propose that the delayed formalin fixation of resection specimen allows cell cycle activities to continue for a long time, up to many hours, and that this leads to an arrest of mitoses in metaphase where they are readily identified by the pathologist. We propose that the mitotic figure count in the rapidly fixed biopsy cores better represent the tumor biology and should be used as a basis for chemotherapy therapeutic decisions.

FTO genotype is associated with phenotypic variability of body mass index.

There is evidence across several species for genetic control of phenotypic variation of complex traits, such that the variance among phenotypes is genotype dependent. Understanding genetic control of variability is important in evolutionary biology, agricultural selection programmes and human medicine, yet for complex traits, no individual genetic variants associated with variance, as opposed to the mean, have been identified. Here we perform a meta-analysis of genome-wide association studies of phenotypic variation using ∼170,000 samples on height and body mass index (BMI) in human populations. We report evidence that the single nucleotide polymorphism (SNP) rs7202116 at the FTO gene locus, which is known to be associated with obesity (as measured by mean BMI for each rs7202116 genotype), is also associated with phenotypic variability. We show that the results are not due to scale effects or other artefacts, and find no other experiment-wise significant evidence for effects on variability, either at loci other than FTO for BMI or at any locus for height. The difference in variance for BMI among individuals with opposite homozygous genotypes at the FTO locus is approximately 7%, corresponding to a difference of ∼0.5 kilograms in the standard deviation of weight. Our results indicate that genetic variants can be discovered that are associated with variability, and that between-person variability in obesity can partly be explained by the genotype at the FTO locus. The results are consistent with reported FTO by environment interactions for BMI, possibly mediated by DNA methylation. Our BMI results for other SNPs and our height results for all SNPs suggest that most genetic variants, including those that influence mean height or mean BMI, are not associated with phenotypic variance, or that their effects on variability are too small to detect even with samples sizes greater than 100,000.

Epigenetic regulation of the bacterial cell cycle.

N(6)-methyl-adenines can serve as epigenetic signals for interactions between regulatory DNA sequences and regulatory proteins that control cellular functions, such as the initiation of chromosome replication or the expression of specific genes. Several of these genes encode master regulators of the bacterial cell cycle. DNA adenine methylation is mediated by Dam in gamma-proteobacteria and by CcrM in alpha-proteobacteria. A major difference between them is that CcrM is cell cycle regulated, while Dam is active throughout the cell cycle. In alpha-proteobacteria, GANTC sites can remain hemi-methylated for a significant period of the cell cycle, depending on their location on the chromosome. In gamma-proteobacteria, most GATC sites are only transiently hemi-methylated, except regulatory GATC sites that are protected from Dam methylation by specific DNA-binding proteins.

Histones trigger sterile inflammation by activating the NLRP3 inflammasome.

Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll-like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)-1β and IL-18. Here, we report that histones released from necrotic cells induce IL-1β secretion in an NLRP3-ASC-caspase-1-dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone-induced IL-1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone-neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell-derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL-1β secretion via oxidative stress.

Un guide pratique au traitement des gliomes [A practical guide for the management of gliomas]

The management of gliomas in daily clinical practice is challenging. It requires a multidisciplinary and coordinated approach involving neurosurgery, radiotherapy and, finally, chemotherapy. Important progress has been made during the last years with the introduction of a combined treatment associating standard radiotherapy with concomitant chemotherapy using temozolomide, a novel alkylating agent. For the first time in many years a new treatment strategy translated into a significant prolongation of survival. In parallel, molecular markers (e.g. loss of heterozygosity on chromosomes 1p and 19q or methylation of the methyl-guanine methyl transferase [MGMT] gene promoter) allowed for identification of distinct subtypes of glioma or prediction of treatment response. In this "Practical Guide", we describe the daily practice and aim at answering some common questions in the management of patients suffering from glioblastoma, astrocytoma, oligodendroglioma and low grade glioma. The therapeutic options presented here are based on evidences from the literature. In the absence of documented evidence, the empirical choices from our local practice are explained and justified.