Faecal microbiota in dogs with multicentric lymphoma

Malignant lymphoma B-cell type is the most common canine haematopoietic malignancy. Changes in intestinal microbiota have been implicated in few types of cancer in humans. The aim of this prospective and case-control study was to determine differences in faecal microbiota between healthy control dogs and dogs with multicentric lymphoma. Twelve dogs affected by multicentric, B-cell, stage III-IV lymphoma, and 21 healthy dogs were enrolled in the study. For each dog, faecal samples were analysed by Illumina sequencing of 16S rRNA genes and quantitative PCR (qPCR) for selected bacterial groups. Alpha diversity was significant lower in lymphoma dogs. Principal coordinate analysis plots showed different microbial clustering (P = .001) and linear discriminant analysis effect size revealed 28 differentially abundant bacterial groups in lymphoma and control dogs. The qPCR analysis showed significant lower abundance of Faecalibacterium spp. (q < .001), Fusobacterium spp. (q = .032), and Turicibacter spp. (q = .043) in dogs with lymphoma compared with control dogs. On the contrary, Streptococcus spp. was significantly higher in dogs with lymphoma (q = .041). The dysbiosis index was significantly higher (P < .0001) in dogs with lymphoma. In conclusion, both sequencing and qPCR analyses provided a global overview of faecal microbial communities and showed significant differences in the microbial communities of dogs presenting with multicentric lymphoma compared with healthy control dogs.dog

The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease.

Recent molecular studies have revealed a highly complex bacterial assembly in the canine intestinal tract. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders. Fecal samples from healthy dogs (n = 32), dogs with acute non-hemorrhagic diarrhea (NHD; n = 12), dogs with acute hemorrhagic diarrhea (AHD; n = 13), and dogs with active (n = 9) and therapeutically controlled idiopathic IBD (n = 10) were analyzed by 454-pyrosequencing of the 16S rRNA gene and qPCR assays. Dogs with acute diarrhea, especially those with AHD, had the most profound alterations in their microbiome, as significant separations were observed on PCoA plots of unweighted Unifrac distances. Dogs with AHD had significant decreases in Blautia, Ruminococcaceae including Faecalibacterium, and Turicibacter spp., and significant increases in genus Sutterella and Clostridium perfringens when compared to healthy dogs. No significant separation on PCoA plots was observed for the dogs with IBD. Faecalibacterium spp. and Fusobacteria were, however, decreased in the dogs with clinically active IBD, but increased during time periods of clinically insignificant IBD, as defined by a clinical IBD activity index (CIBDAI). Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with various GI disorders. The observed changes in the microbiome differed between acute and chronic disease states. The bacterial groups that were commonly decreased during diarrhea are considered to be important short-chain fatty acid producers and may be important for canine intestinal health. Future studies should correlate these observed phylogenetic differences with functional changes in the intestinal microbiome of dogs with defined disease phenotypes

Carbohydrate-Free Peach (Prunus persica) and Plum (Prunus salicina) [corrected] Juice Affects Fecal Microbial Ecology in an Obese Animal Model.

BACKGROUND Growing evidence shows the potential of nutritional interventions to treat obesity but most investigations have utilized non-digestible carbohydrates only. Peach and plum contain high amounts of polyphenols, compounds with demonstrated anti-obesity effects. The underlying process of successfully treating obesity using polyphenols may involve an alteration of the intestinal microbiota. However, this phenomenon is not well understood. METHODOLOGY/PRINCIPAL FINDINGS Obese Zucker rats were assigned to three groups (peach, plum, and control, n = 10 each), wild-type group was named lean (n = 10). Carbohydrates in the fruit juices were eliminated using enzymatic hydrolysis. Fecal samples were obtained after 11 weeks of fruit or control juice administration. Real-time PCR and 454-pyrosequencing were used to evaluate changes in fecal microbiota. Over 1,500 different Operational Taxonomic Units at 97% similarity were detected in all rats. Several bacterial groups (e.g. Lactobacillus and members of Ruminococcacea) were found to be more abundant in the peach but especially in the plum group (plum juice contained 3 times more total polyphenolics compared to peach juice). Principal coordinate analysis based on Unifrac-based unweighted distance matrices revealed a distinct separation between the microbiota of control and treatment groups. These changes in fecal microbiota occurred simultaneously with differences in fecal short-chain acids concentrations between the control and treatment groups as well as a significant decrease in body weight in the plum group. CONCLUSIONS This study suggests that consumption of carbohydrate-free peach and plum juice has the potential to modify fecal microbial ecology in an obese animal model. The separate contribution of polyphenols and non-polyphenols compounds (vitamins and minerals) to the observed changes is unknown

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement

Detection and Characterization of Oncogene Mutations in Preneoplastic and Early Neoplastic Lesions

While it has been nearly 30 years since its discovery, the ras family of genes has not yet lost its impact on basic and clinical oncology. These genes remain central to the field of molecular oncology as tools for investigating carcinogenesis and oncogenic signaling, as powerful biomarkers for the identification of those who have or are at high risk of developing cancer, and as oncogene targets for the design and development of new chemotherapeutic drugs. Mutational activation of the K-RAS proto-oncogene is an early event in the development and progression of the colorectal, pancreatic, and lung cancers that are the major causes of cancer death in the world. The presence of point mutational "hot spots" at sites necessary for the activation of this proto-oncogene has led to the development of a number of highly sensitive PCR-based methods that are feasible for the early detection of K-RAS oncogene mutations in the clinical setting. In light of these facts, mutation at the K-RAS oncogene has the potential to serve as a useful biomarker in the early diagnosis and risk assessment of cancers with oncogenic ras signaling. This chapter describes a highly sensitive method for detecting mutant K-RAS, enriched PCR, and its application to early detection of alterations in this oncogene in preneoplastic and early neoplastic lesions of the colon and rectum

Fecal Microbial and Metabolic Profiles in Dogs With Acute Diarrhea Receiving Either Fecal Microbiota Transplantation or Oral Metronidazole

The aim was to characterize differences in fecal consistency, and fecal microbiota and metabolome profiles in dogs with acute diarrhea (AD) treated with either fecal microbiota transplantation as enema (FMT;n = 11) or oral metronidazole (MET;n = 7) for 7 days. On days 0, 7, and 28 fecal samples were obtained. Fecal samples from healthy dogs (HC;n = 14) were used for comparison. Samples were analyzed by the previously validated qPCR based canine Dysbiosis Index (DI;increased values indicate microbiota dysbiosis) and 16S rRNA gene sequencing. The fecal metabolome was analyzed using a previously validated targeted canine assay for fecal unconjugated bile acids, and untargeted metabolomics. Fecal consistency improved significantly in dogs treated with FMT and MET by day 7 and day 28 (p < 0.01) compared to day 0. However, on day 28 fecal consistency was significantly better in FMT compared to MET (p = 0.040). At day 0, dogs with AD had an altered microbiota indicated by significantly increased DI, decreased alpha-diversity, and altered beta-diversity. In the FMT group, the DI decreased over time, while MET led to a significant increase in the dysbiosis index at day 7 and 28 compared to FMT. Sequencing data revealed that in FMT microbial diversity and beta-diversity was similar to HC at day 28, while in MET these parameters were still significantly different from HC. In dogs treated with FMT, a decrease in cholic acid and the percentage of primary bile acids was observed, whereas treatment with metronidazole led to an increase in cholic acid at day 7 and an increase in percentage of primary bile acids over time. Based on untargeted metabolomics, dogs with AD had an altered fecal metabolome compared to HC. Dogs treated with FMT clustered closer to HC at day 28, while dogs treated with MET did not. In this pilot study, dogs with AD had significant differences in fecal microbiota and metabolome profiles. Dogs treated with MET still had altered microbial and metabolic profiles at day 28 compared to dogs treated with FMT or healthy dogs

Helicobacter pylori Infection Promotes Methylation and Silencing of Trefoil Factor 2, Leading to Gastric Tumor Development in Mice and Humans

Background & Aims Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. Methods We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130[superscript F/F] mouse model of gastric cancer. Results In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130[superscript F/F]/Tff2[superscript −/−] mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130[superscript F/F]controls. The fundus of gp130[superscript F/F]/Tff2[superscript −/−] mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. Conclusions TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.National Health and Medical Research Council (Australia

Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression

Abstract Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS)-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema. Methods Mice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle) by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP) activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages. Results Quercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype. Conclusions Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.http://deepblue.lib.umich.edu/bitstream/2027.42/78260/1/1465-9921-11-131.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78260/2/1465-9921-11-131.pdfPeer Reviewe