Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

Transcription is required for establishment of germline methylation marks at imprinted genes.

Genomic imprinting requires the differential marking by DNA methylation of genes in male and female gametes. In the female germline, acquisition of methylation imprint marks depends upon the de novo methyltransferase Dnmt3a and its cofactor Dnmt3L, but the reasons why specific sequences are targets for Dnmt3a and Dnmt3L are still poorly understood. Here, we investigate the role of transcription in establishing maternal germline methylation marks. We show that at the Gnas locus, truncating transcripts from the furthest upstream Nesp promoter disrupts oocyte-derived methylation of the differentially methylated regions (DMRs). Transcription through DMRs in oocytes is not restricted to this locus but occurs across the prospective DMRs at many other maternally marked imprinted domains, suggesting a common requirement for transcription events. The transcripts implicated here in gametic methylation are protein-coding, in contrast to the noncoding antisense transcripts involved in the monoallelic silencing of imprinted genes in somatic tissues, although they often initiate from alternative promoters in oocytes. We propose that transcription is a third essential component of the de novo methylation system, which includes optimal CpG spacing and histone modifications, and may be required to create or maintain open chromatin domains to allow the methylation complex access to its preferred targets.

Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches.

Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position.

Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster.

There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs.

Aromatase inhibitors for treatment of advanced breast cancer in postmenopausal women.

BACKGROUND: Endocrine therapy removes the influence of oestrogen on breast cancer cells and so hormonal treatments such as tamoxifen, megestrol acetate and medroxyprogesterone acetate have been in use for many years for advanced breast cancer. Aromatase inhibitors (AIs) inhibit oestrogen synthesis in the peripheral tissues and have a similar tumour-regressing effect to other endocrine treatments. Aminoglutethimide was the first AI in clinical use and now the third generation AIs, anastrozole, exemestane and letrozole, are in current use. Randomised trial evidence on response rates and side effects of these drugs is still limited. OBJECTIVES: To compare AIs to other endocrine therapy in the treatment of advanced breast cancer in postmenopausal women. SEARCH STRATEGY: For this update, the Cochrane Breast Cancer Group Specialised Register and the Cochrane Central Register of Controlled Trials (CENTRAL) and relevant conference proceedings were searched (to 30 June 2008). SELECTION CRITERIA: Randomised controlled trials in postmenopausal women comparing the effects of any AI versus other endocrine therapy, no endocrine therapy, or a different AI in the treatment of advanced (metastatic) breast cancer. Non-English language publications, comparisons of the same AI at different doses, AIs used as neoadjuvant treatment, or outcomes not related to tumour response were excluded. DATA COLLECTION AND ANALYSIS: Data from published trials were extracted independently by two review authors and cross-checked by a third. Hazard ratios (HR) were derived for analysis of time-to-event outcomes (overall and progression-free survival). Odds ratios (OR) were derived for objective response, clinical benefit, and toxicity. MAIN RESULTS: Thirty-seven trials were identified, 31 of which were included in the main analysis of any AI versus any other treatment (11,403 women). No trials were excluded due to inadequate allocation concealment. The pooled estimate showed a significant survival benefit for treatment with an AI over other endocrine therapies (HR 0.90, 95% CI 0.84 to 0.97). A subgroup analysis of the three commonly prescribed AIs (anastrozole, exemestane, letrozole) also showed a similar survival benefit (HR 0.88, 95% CI 0.80 to 0.96). There were very limited data to compare one AI with a different AI, but these suggested an advantage for letrozole over anastrozole.AIs have a different toxicity profile to other endocrine therapies. For those currently prescribed, and for all AIs combined, they had similar levels of hot flushes and arthralgia; increased risks of rash, nausea, diarrhoea and vomiting; but a 71% decreased risk of vaginal bleeding and 47% decrease in thromboembolic events compared with other endocrine therapies. AUTHORS' CONCLUSIONS: In women with advanced (metastatic) breast cancer, aromatase inhibitors including those in current clinical use show a survival benefit when compared to other endocrine therapy.

Phenotypic and molecular characterisation of Brucella isolates from marine mammals.

BACKGROUND: Bacteria of the genus Brucella are the causative organisms of brucellosis in animals and man. Previous characterisation of Brucella strains originating from marine mammals showed them to be distinct from the terrestrial species and likely to comprise one or more new taxa. Recently two new species comprising Brucella isolates from marine mammals, B. pinnipedialis and B. ceti, were validly published. Here we report on an extensive study of the molecular and phenotypic characteristics of marine mammal Brucella isolates and on how these characteristics relate to the newly described species. RESULTS: In this study, 102 isolates of Brucella originating from eleven species of marine mammals were characterised. Results obtained by analysis using the Infrequent Restriction Site (IRS)-Derivative PCR, PCR-RFLP of outer membrane protein genes (omp) and IS711 fingerprint profiles showed good consistency with isolates originating from cetaceans, corresponding to B. ceti, falling into two clusters. These correspond to isolates with either dolphins or porpoises as their preferred host. Isolates originating predominantly from seals, and corresponding to B. pinnipedialis, cluster separately on the basis of IS711 fingerprinting and other molecular approaches and can be further subdivided, with isolates from hooded seals comprising a distinct group. There was little correlation between phenotypic characteristics used in classical Brucella biotyping and these groups. CONCLUSION: Molecular approaches are clearly valuable in the division of marine mammal Brucella into subtypes that correlate with apparent ecological divisions, whereas conventional bioyping is of less value. The data presented here confirm that there are significant subtypes within the newly described marine mammal Brucella species and add to a body of evidence that could lead to the recognition of additional species or sub-species within this group.

Contribution of TNFSF15 gene variants to Crohn's disease susceptibility confirmed in UK population.

BACKGROUND: Identification of Crohn's disease (CD)-associated genetic variants is key to understanding pathogenic pathways underlying disease susceptibility. Recent reports of an association between TNFSF15 variants and CD have been modestly replicated in European populations, suggesting heterogeneity at this locus with stronger CD association in Japanese than European populations. METHODS: We investigated the association between variants in TNFSF15 and CD in 756 CD patients and 636 controls. Disease subphenotype associations were also investigated. RESULTS: TNFSF15 single nucleotide polymorphism (SNP) variants were associated with CD in our panel with peak odds ratio (OR) 1.2 (95% confidence interval [CI] 1.01-1.41) P = 0.033. The presence of a risk haplotype was replicated for the first time in a European population (frequency 67% in cases and 61% in controls) OR = 1.44 (95% CI 1.23-1.68) P = 0.00012. This result mirrors the UK panel in the index study (Yamazaki et al [2005] Hum Mol Genet 14:3499-3506) but is less significant than that reported in Japanese populations. There was no evidence of association with any individual CD subphenotype. CONCLUSIONS: Variants in TNFSF15 contribute to overall CD susceptibility in European populations, although to a lesser extent than that seen in the Japanese. Further studies to define the precise disease-causing variants as well as targeted functional studies are now required in human CD as TNFSF15 is a potential target for biological therapies.

Antagonists of the human adenosine A2A receptor. Part 3: Design and synthesis of pyrazolo[3,4-d]pyrimidines, pyrrolo[2,3-d]pyrimidines and 6-arylpurines.

A series of pyrazolo[3,4-d]pyrimidine, pyrrolo[2,3-d]pyrimidine and 6-arylpurine adenosine A(2A) antagonists is described. Many examples were highly selective against the human A(1) receptor sub-type and were active in an in vivo model of Parkinson's disease.

Antagonists of the human adenosine A2A receptor. Part 2: Design and synthesis of 4-arylthieno[3,2-d]pyrimidine derivatives.

We describe herein the discovery and development of a series of 4-arylthieno[3,2-d]pyrimidines which are potent adenosine A(2A) receptor antagonists. These novel compounds show high degrees of selectivity against the human A(1), A(2B) and A(3) receptor sub-types. Moreover, a number of these compounds show promising activity in vivo, suggesting potential utility in the treatment of Parkinson's disease.

Antagonists of the human adenosine A2A receptor. Part 1: Discovery and synthesis of thieno[3,2-d]pyrimidine-4-methanone derivatives.

The (-)-(11R,2'S)-enantiomer of the antimalarial drug mefloquine has been found to be a reasonably potent and moderately selective adenosine A(2A) receptor antagonist. Further investigation of this compound has led to the discovery of a series of keto-aryl thieno[3,2-d]pyrimidine derivatives, which are potent and selective antagonists of the adenosine A(2A) receptor. These derivatives show selectivity against the A(1) receptor. Furthermore, some of these compounds have been shown to have in vivo activity in a commonly used model, suggesting the potential for the treatment of Parkinson's disease.

Brucella ceti Infection in a Common Minke Whale ( Balaenoptera acutorostrata ) with Associated Pathology.

There are three major lineages of marine mammal strains of Brucella spp.: Brucella ceti ST23, found predominantly in porpoises; B. ceti ST26, in pelagic delphinids and ziphiids; and Brucella pinnipedialis ST24/25, predominantly in seals. The isolation of Brucella spp. in mysticetes has been described only in common minke whales ( Balaenoptera acutorostrata ) in Norway and Scotland. We report a third case of Brucella infection and isolation in a minke whale associated with a large abscess. In contrast to the two previous reports that involved isolates of B. pinnipedialis ST24 or the porpoise-associated B. ceti complex ST23, this case was associated with the dolphin-associated B. ceti ST26. Thus, minke whales can be infected naturally with members of all the distinct major lineages of Brucella associated with marine mammals. This report is unique in that the B. ceti ST26 did not originate from a pelagic delphinid or a beaked whale.

Characterisation of North American Brucella isolates from marine mammals.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

Serologic survey for Brucella spp., phocid herpesvirus-1, phocid herpesvirus-2, and phocine distemper virus in harbor seals from Alaska, 1976-1999.

Harbor seals (Phoca vitulina richardsi) were captured in the coastal regions of Southeast Alaska, Gulf of Alaska, Prince William Sound (PWS), and Kodiak Island during 1976-1999. Blood was collected from 286 seals. Sera were tested for evidence of exposure to Brucella spp., phocid herpesvirus-1 (PhoHV-1), phocid herpesvirus-2 (PhHV-2), and phocine distemper virus (PDV). Antibody prevalence rates were 46% (46/100) for Brucella spp., 93% (225/243) for PhoHV-1, 0% (0/286) for PhHV-2, and 1% (2/160) for PDV. Antibody prevalence for Brucella spp. was directly related to host age. Antibody prevalence for PhoHV-1 was higher in PWS as compared to the other three regions. No evidence of mortality attributable to these four agents was observed during the course of this study. Based on the results of this survey, none of these agents is considered a significant mortality factor in harbor seals from the four regions of coastal Alaska included in the study.

Meningoencephalitis and arthritis associated with Brucella ceti in a short-beaked common dolphin (Delphinus delphis).

Brucella species infection in marine mammal species has been reported to have a global distribution. In 2007, the description of Brucella ceti was published and formally adopted for those isolates originating from cetaceans and pathologic lesions similar to those seen in terrestrial mammals infected with Brucella spp. have been associated with its isolation. Brucella ceti infection specific to the central nervous system has been described in two species of cetacean: striped dolphins (Stenella coeruleoalba) in Europe and Costa Rica and an Atlantic white-sided dolphin (Lagenorhynchus acutus) in the UK. We describe the first report, to our knowledge, of B. ceti-associated meningitis and arthritis in a third species, the short-beaked common dolphin (Delphinus delphis), in an animal that stranded in the UK.

Analysis of volatile organic compounds of bacterial origin in chronic gastrointestinal diseases.

BACKGROUND: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. METHODS: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn's disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40°C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography-mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. RESULTS: Crohn's disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. CONCLUSIONS: The abundance of a number of VOCs in feces differs markedly between Crohn's disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

Mucosal genome-wide methylation changes in inflammatory bowel disease.

BACKGROUND: DNA methylation constitutes a key epigenetic mechanism by which cells regulate gene transcription. Among its roles are the dynamic regulation of gene expression, for example, as part of an evolving immune response, and cell differentiation in specialized tissues. Here our aim was to study the impact of differences in methylation patterns in the intestine with regard to inflammatory bowel disease (IBD) susceptibility and activity. METHODS: Having extracted DNA from rectal biopsies, we conducted genome-wide methylation profiling using the HumanMethylation27 BeadChip microarray to identify genes showing evidence of differential methylation between cases of ulcerative colitis and Crohn's disease and healthy controls. Selected methylation signals were validated in an independent replication panel by pyrosequencing. Correlation with gene expression was sought by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: Multiple genes showed significant evidence of differential methylation, several appearing in both ulcerative colitis and Crohn's disease comparisons including THRAP2, FANCC, GBGT1, DOK2, TNFSF4, TNFSF12, and FUT7. Many more than expected by chance overlapped with genes previously implicated as playing a role in IBD susceptibility in genome-wide association scans, including CARD9, ICAM3, and IL8RB (P < 0.001). Correlation between methylation and gene expression was identified for selected transcripts. CONCLUSIONS: Consistent differences in DNA methylation between IBD cases and controls at regulatory sites within these genes suggest that their altered transcription contributes to IBD pathogenesis.

Diversity and distribution of sulphate-reducing bacteria in human faeces from healthy subjects and patients with inflammatory bowel disease.

The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohn's disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B. wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.

Is the abundance of Faecalibacterium prausnitzii relevant to Crohn's disease?

Reports that bacteria within the Firmicutes phylum, especially the species Faecalibacterium prausnitzii, are less abundant in Crohn's disease (CD) patients and supernatants from cultures of this bacterium are anti-inflammatory prompted the investigation of the possible correlations between the abundance of F. prausnitzii and the response to treatment in patients with gut diseases and healthy controls. In a randomized, double-blind trial, faeces were collected from healthy volunteers, and from patients with active CD, ulcerative colitis (UC) and irritable bowel syndrome before and after treatment. The levels of F. prausnitzii DNA in faecal suspensions were determined by PCR. Treatment by an elemental diet was effective, resulting in decreases in both the Harvey and Bradshaw index (P<0.001) and the concentrations of serum C-reactive protein (P<0.05). The total levels of F. prausnitzii in faecal samples from CD patients at presentation were lower than those in the other groups both before and after the treatment. There was no correlation between F. prausnitzii abundance and the severity of CD before treatment. Clinical improvement unexpectedly correlated with a significant decrease in the abundance of F. prausnitzii, especially the A2-165 subgroup (P<0.05). Our data suggest that a paucity of F. prausnitzii in the gastrointestinal microbial communities is likely to be a minor aetiological factor in CD: recovery following elemental diet is attributed to lower levels of gut flora.

Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5.

Mouse ES cells can differentiate into all three germ layers of the embryo but are generally excluded from the trophoblast lineage. Here we show that ES cells deficient in DNA methylation can differentiate efficiently into trophoblast derivatives. In a genome-wide screen we identified the transcription factor Elf5 as methylated and repressed in ES cells, and hypomethylated and expressed in TS and methylation-deficient ES cells. Elf5 creates a positive-feedback loop with the TS cell determinants Cdx2 and Eomes that is restricted to the trophoblast lineage by epigenetic regulation of Elf5. Importantly, the late-acting function of Elf5 allows initial plasticity and regulation in the early blastocyst. Thus, Elf5 functions as a gatekeeper, downstream of initial lineage determination, to reinforce commitment to the trophoblast lineage or to abort this pathway in epiblast cells. This epigenetic restriction of cell lineage fate provides a molecular mechanism for Waddington's concept of canalization of developmental pathways.