M3 Receptor Pathway Stimulates Rapid Transcription of the CB1 Receptor Activation through Calcium Signalling and the CNR1 Gene Promoter.

In this study, we have investigated a possible mechanism that enables CB1/M3 receptor cross-talk, using SH-SY5Y cells as a model system. Our results show that M3 receptor activation initiates signaling that rapidly upregulates the CNR1 gene, resulting in a greatly potentiated CB1 receptor response to agonists. Calcium homeostasis plays an essential intermediary role in this functional CB1/M3 receptor cross-talk. We show that M3 receptor-triggered calcium release greatly increases CB1 receptor expression via both transcriptional and translational activity, by enhancing CNR1 promoter activity. The co-expression of M3 and CB1 receptors in brain areas such as the nucleus accumbens and amygdala support the hypothesis that the altered synaptic plasticity observed after exposure to cannabinoids involves cross-talk with the M3 receptor subtype. In this context, M3 receptors and their interaction with the cannabinoid system at the transcriptional level represent a potential pharmacogenomic target not only for the develop of new drugs for addressing addiction and tolerance. but also to understand the mechanisms underpinning response stratification to cannabinoids.

Disease-associated polymorphisms within the conserved ECR1 enhancer differentially regulate the tissue-specific activity of the cannabinoid-1 receptor gene promoter; implications for cannabinoid pharmacogenetics.

Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Toward addressing the hypothesis that genetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we characterized the effects of disease-associated allelic variation within a conserved regulatory sequence (ECR1) in CNR1 intron 2 that had previously been shown to modulate cannabinoid response, alcohol intake, and anxiety-like behavior. We used primary cell analysis of reporters carrying different allelic variants of the human ECR1 and found that human-specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in primary hippocampal cells than did the ancestral T-allele and demonstrated a differential response to CB1 agonism. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity and evidence that the ancestral t-allele variant of ECR1 interacted with higher affinity with the insulator binding factor CTCF. The cell-specific approaches used in our study represent an important step in gaining a mechanistic understanding of the roles of noncoding polymorphic variation in disease and in the increasingly important field of cannabinoid pharmacogenetics.

The noncoding human genome and the future of personalised medicine.

Non-coding cis-regulatory sequences act as the 'eyes' of the genome and their role is to perceive, organise and relay cellular communication information to RNA polymerase II at gene promoters. The evolution of these sequences, that include enhancers, silencers, insulators and promoters, has progressed in multicellular organisms to the extent that cis-regulatory sequences make up as much as 10% of the human genome. Parallel evidence suggests that 75% of polymorphisms associated with heritable disease occur within predicted cis-regulatory sequences that effectively alter the 'perception' of cis-regulatory sequences or render them blind to cell communication cues. Cis-regulatory sequences also act as major functional targets of epigenetic modification thus representing an important conduit through which changes in DNA-methylation affects disease susceptibility. The objectives of the current review are (1) to describe what has been learned about identifying and characterising cis-regulatory sequences since the sequencing of the human genome; (2) to discuss their role in interpreting cell signalling pathways pathways; and (3) outline how this role may be altered by polymorphisms and epigenetic changes. We argue that the importance of the cis-regulatory genome for the interpretation of cellular communication pathways cannot be overstated and understanding its role in health and disease will be critical for the future development of personalised medicine.

Atypical presentation of intravascular leiomyomatosis mimicking advanced uterine sarcoma: modified laterally extended endopelvic resection with preservation of pelvic neural structures.

Intravascular leiomyomatosis is a rare, benign tumour of myometrial smooth muscle. Despite being non-invasive, these tumours can proliferate aggressively within vascular structures including pelvic vessels, the vena cava and the heart. We discuss a 77-year-old woman presenting with a 9 cm uterine mass extending into the right adnexa and ovarian vein. Following hysteroscopic biopsy, palliative radical surgical resection was performed for suspected stage IV leiomyosarcoma. Tumour extension into the pelvic sidewall and obturator fossa indicated a modified laterally extended endopelvic resection combined with skeletonisation and preservation of the pelvic neurovasculature, ultimately providing a curative procedure with minimal functional neurological morbidity. We present this unusual case to assist in the development of a consensus for optimal case management where formal guidelines are not yet available. We summarise current understanding of intravascular leiomyomatosis and highlight the value of advanced surgical techniques using knowledge of complex ontogenetic and pelvic neuroanatomy in its management.

The flagellin of candidate live biotherapeutic Enterococcus gallinarum MRx0518 is a potent immunostimulant.

Many links between gut microbiota and disease development have been established in recent years, with particular bacterial strains emerging as potential therapeutics rather than causative agents. In this study we describe the immunostimulatory properties of Enterococcus gallinarum MRx0518, a candidate live biotherapeutic with proven anti-tumorigenic efficacy. Here we demonstrate that strain MRx0518 elicits a strong pro-inflammatory response in key components of the innate immune system but also in intestinal epithelial cells. Using a flagellin knock-out derivative and purified recombinant protein, MRx0518 flagellin was shown to be a TLR5 and NF-κB activator in reporter cells and an inducer of IL-8 production by HT29-MTX cells. E. gallinarum flagellin proteins display a high level of sequence diversity and the flagellin produced by MRx0518 was shown to be more potent than flagellin from E. gallinarum DSM100110. Collectively, these data infer that flagellin may play a role in the therapeutic properties of E. gallinarum MRx0518.

Bifidobacterium breve MRx0004 protects against airway inflammation in a severe asthma model by suppressing both neutrophil and eosinophil lung infiltration.

Asthma is a phenotypically heterogeneous disease. In severe asthma, airway inflammation can be predominantly eosinophilic, neutrophilic, or mixed. Only a limited number of drug candidates are in development to address this unmet clinical need. Live biotherapeutics derived from the gut microbiota are a promising new therapeutic area. MRx0004 is a commensal Bifidobacterium breve strain isolated from the microbiota of a healthy human. The strain was tested prophylactically and therapeutically by oral gavage in a house dust mite mouse model of severe asthma. A strong reduction of neutrophil and eosinophil infiltration was observed in lung bronchoalveolar lavage fluid following MRx0004 treatment. Peribronchiolar and perivascular immunopathology was also reduced. MRx0004 increased lung CD4+CD44+ cells and CD4+FoxP3+ cells and decreased activated CD11b+ dendritic cells. Cytokine analysis of lung tissue revealed reductions of pro-inflammatory cytokines and chemokines involved in neutrophil migration. In comparison, anti-IL-17 antibody treatment effectively reduced neutrophilic infiltration and increased CD4+FoxP3+ cells, but it induced lung eosinophilia and did not decrease histopathology scores. We have demonstrated that MRx0004, a microbiota-derived bacterial strain, can reduce both neutrophilic and eosinophilic infiltration in a mouse model of severe asthma. This novel therapeutic is a promising next-generation drug for management of severe asthma.

Determining Epigenetic Targets: A Beginner's Guide to Identifying Genome Functionality Through Database Analysis.

There can now be little doubt that the cis-regulatory genome represents the largest information source within the human genome essential for health. In addition to containing up to five times more information than the coding genome, the cis-regulatory genome also acts as a major reservoir of disease-associated polymorphic variation. The cis-regulatory genome, which is comprised of enhancers, silencers, promoters, and insulators, also acts as a major functional target for epigenetic modification including DNA methylation and chromatin modifications. These epigenetic modifications impact the ability of cis-regulatory sequences to maintain tissue-specific and inducible expression of genes that preserve health. There has been limited ability to identify and characterize the functional components of this huge and largely misunderstood part of the human genome that, for decades, was ignored as "Junk" DNA. In an attempt to address this deficit, the current chapter will first describe methods of identifying and characterizing functional elements of the cis-regulatory genome at a genome-wide level using databases such as ENCODE, the UCSC browser, and NCBI. We will then explore the databases on the UCSC genome browser, which provides access to DNA methylation and chromatin modification datasets. Finally, we will describe how we can superimpose the huge volume of study data contained in the NCBI archives onto that contained within the UCSC browser in order to glean relevant in vivo study data for any locus within the genome. An ability to access and utilize these information sources will become essential to informing the future design of experiments and subsequent determination of the role of epigenetics in health and disease and will form a critical step in our development of personalized medicine.

Functional effects of polymorphisms on glucocorticoid receptor modulation of human anxiogenic substance-P gene promoter activity in primary amygdala neurones.

Expression or introduction of the neuropeptide substance-P (SP; encoded by the TAC1 gene in humans and Tac1 in rodents) in the amygdala induces anxiety related behaviour in rodents. In addition, pharmacological antagonism of the main receptor of SP in humans; NK1, is anxiolytic. In the current study, we show that the Tac1 locus is up-regulated in primary rat amygdala neurones in response to activation of the glucocorticoid receptor (GR); a classic component of the stress response. Using a combination of bioinformatics, electrophoretic mobility shift assays (EMSA) and reporter plasmid magnetofection into rat primary amygdala neurones we identified a highly conserved GR response sequence (2GR) in the human TAC1 promoter that binds GR in response to dexamethasone (Dex) or forskolin. We also identified a second GR binding site in the human promoter that was polymorphic and whose T-allele is only found in Japanese and Chinese populations. We present evidence that the T-allele of SNPGR increases the activity of the TAC1 promoter through de-sequestration or de-repression of 2GR. The identification of Dex/forskolin response elements in the TAC1 promoter in amygdala neurones suggests a possible link in the chain of molecular events connecting GR activation and anxiety. In addition, the discovery of a SNP which can alter this response may have implications for our understanding of the role of regulatory variation in susceptibility to stress in specific populations.

Understanding the Dynamics of Gene Regulatory Systems; Characterisation and Clinical Relevance of cis-Regulatory Polymorphisms.

Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRSs that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics and experimental techniques that allow for the identification of CRSs building on recent advances by the ENCODE consortium. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and stratification whilst providing novel directions for the development of personalised medicine.