Using gene expression data to identify certain gastro-intestinal diseases

BACKGROUND: Inflammatory bowel diseases, ulcerative colitis and Crohn’s disease are considered to be of autoimmune origin, but the etiology of irritable bowel syndrome remains elusive. Furthermore, classifying patients into irritable bowel syndrome and inflammatory bowel diseases can be difficult without invasive testing and holds important treatment implications. Our aim was to assess the ability of gene expression profiling in blood to differentiate among these subject groups. METHODS: Transcript levels of a total of 45 genes in blood were determined by quantitative real-time polymerase chain reaction (RT-PCR). We applied three separate analytic approaches; one utilized a scoring system derived from combinations of ratios of expression levels of two genes and two different support vector machines. RESULTS: All methods discriminated different subject cohorts, irritable bowel syndrome from control, inflammatory bowel disease from control, irritable bowel syndrome from inflammatory bowel disease, and ulcerative colitis from Crohn’s disease, with high degrees of sensitivity and specificity. CONCLUSIONS: These results suggest these approaches may provide clinically useful prediction of the presence of these gastro-intestinal diseases and syndromes

Systemic sclerosis is associated with specific alterations in gastrointestinal microbiota in two independent cohorts.

ObjectiveTo compare faecal microbial composition in patients with systemic sclerosis (SSc) from 2 independent cohorts with controls and to determine whether certain genera are associated with SSc-gastrointestinal tract (GIT) symptoms.DesignAdult patients with SSc from the University of California, Los Angeles (UCLA) and Oslo University Hospital (OUH) and healthy controls participated in this study (1:1:1). All participants provided stool specimens for 16S rRNA sequencing. Linear discriminant analysis effect size demonstrated genera with differential expression in SSc. Differential expression analysis for sequence count data identified specific genera associated with GIT symptoms as assessed by the GIT 2.0 questionnaire.ResultsThe UCLA-SSc and OUH-SSc cohorts were similar in age (52.1 and 60.5 years, respectively), disease duration (median (IQR): 6.6 (2.5-16.4) and 7.0 (1.0-19.2) years, respectively), gender distribution (88% and 71%, respectively), and GIT symptoms (mean (SD) total GIT 2.0 scores of 0.7 (0.6) and 0.6 (0.5), respectively). Principal coordinate analysis illustrated significant microbial community differences between SSc and controls (UCLA: p=0.001; OUH: p=0.002). Patients with SSc had significantly lower levels of commensal genera deemed to protect against inflammation, such as Bacteroides (UCLA and OUH), Faecalibacterium (UCLA), Clostridium (OUH); and significantly higher levels of pathobiont genera, such as Fusobacterium (UCLA), compared with controls. Increased abundance of Clostridium was associated with less severe GIT symptoms in both cohorts.ConclusionsThe present analysis detected specific aberrations in the lower GIT microbiota of patients with SSc from 2 geographically and ethnically distinct cohorts. These findings suggest that GIT dysbiosis may be a pathological feature of the SSc disease state

A large population-based investigation into the genetics of susceptibility to gastrointestinal infections and the link between gastrointestinal infections and mental illness.

Gastrointestinal infections can be life threatening, but not much is known about the host's genetic contribution to susceptibility to gastrointestinal infections or the latter's association with psychiatric disorders. We utilized iPSYCH, a genotyped population-based sample of individuals born between 1981 and 2005 comprising 65,534 unrelated Danish individuals (45,889 diagnosed with mental disorders and 19,645 controls from a random population sample) in which all individuals were linked utilizing nationwide population-based registers to estimate the genetic contribution to susceptibility to gastrointestinal infections, identify genetic variants associated with gastrointestinal infections, and examine the link between gastrointestinal infections and psychiatric and neurodevelopmental disorders. The SNP heritability of susceptibility to gastrointestinal infections ranged from 3.7% to 6.4% on the liability scale. Significant correlations were found between gastrointestinal infections and the combined group of mental disorders (OR = 2.09; 95% CI: 1.82-2.4, P = 1.87 × 10-25). Correlations with autism spectrum disorder, attention deficit hyperactivity disorder, and depression were also significant. We identified a genome-wide significant locus associated with susceptibility to gastrointestinal infections (OR = 1.13; 95% CI: 1.08-1.18, P = 2.9 × 10-8), where the top SNP was an eQTL for the ABO gene. The risk allele was associated with reduced ABO expression, providing, for the first time, genetic evidence to support previous studies linking the O blood group to gastrointestinal infections. This study also highlights the importance of integrative work in genetics, psychiatry, infection, and epidemiology on the road to translational medicine

Diagnostics of inflammatory bowel disease using fecal microbiota: Diagnostic markers and commercial potential

Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2010. Master of applied and commercial biotechnology.Establishing the diagnosis of Inflammatory Bowel Disease (IBD) with its two main sub forms Crohn‟s Disease (CD) and Ulcerative colitis (UC) are based on medical history, clinical evaluation, laboratory tests, endoscopy, radiology and histology. However no gold standard exists. The lack of appropriate diagnostic tools leads to delayed and incorrect treatment of IBD patients. A substantial amount of patients diagnosed as CD are later reclassified as UC and opposite. Also the type of colitis remains unclassified in many patients. In addition, non- IBD patients presenting with similar symptoms as IBD are unnecessarily investigated with invasive tests leading to increased hospitals costs. The cause of IBD is not yet completely described, but most evidence points to a combination of genetic predisposition, immunological factors, environmental triggers, and gastro intestinal (GI) microbes. However, neither the types of microbes responsible for the diseases nor changes in the microbiota as a result of the diseases have been sufficiently identified. The aim of this thesis was to evaluate the potential of using the fecal microbiota for IBD diagnostics. This was achieved through a combination of a literature study, lab study and investigations of the commercial potential including a patent search. The literature study revealed conflicting evidence related to the amounts of bacteria in IBD patients relative to controls. Nevertheless, a majority of the articles agreed in decreased amounts of Clostridia species and increased amounts of Gammaproteobacteria species in the GI microbiota of IBD patients. The lab study comprised an evaluation of a genetic test, GA-map™, commercialized by Genetic Analysis AS (GA). By using variable regions in the 16S rRNA gene, simultaneous detection and identification of multiple bacteria in a complex mixture of DNA is possible. Probes and analytic methods are suitable for several types of diagnostic tests among other IBD. A sequence analysis of fecal samples from 152 IBD patients and 105 non-IBD controls was performed. Significantly a probe detecting increased relative amounts of Proteobacteria and Bacteroidetes species was identified as a new possible diagnostic test for CD patients. A search in European and American patent databases revealed several patents related to IBD diagnostics. Especially important, a patent application from George Mason University comprised claims referring to IBD diagnostics by using the microbial community of the digestive tract and lumen. If issued, this application could influence the freedom to operate to companies focusing on bacterial markers in IBD diagnostics. Few other patents or patent applications from the search query include claims for identification of bacteria in fecal samples. A concluding remark from examining the commercial potential of IBD diagnostics in this thesis is to tailor make a diagnostic test separating IBD from irritating bowel syndrome (IBS), a common functional disease frequently confused with IBD. Based on estimations of price and profit per test, a €7Mill research budget was recommended for the normal case scenario. Finally it is concluded that development of a diagnostic test based on fecal microbiota has a commercial potential within the proposed framework

Gastrointestinal neuromuscular apparatus: An underestimated target of gut microbiota

Over the last few years, the importance of the resident intestinal microbiota in the pathogenesis of several gastro- intestinal diseases has been largely investigated. Growing evidence suggest that microbiota can influence gastro- intestinal motility. The current working hypothesis is that dysbiosis-driven mucosal alterations induce the production of several inflammatory/immune mediators which affect gut neuro-muscular functions. Besides these indirect mucosal-mediated effects, the present review highlights that recent evidence suggests that microbiota can directly affect enteric nerves and smooth muscle cells functions through its metabolic products or bacterial molecular components translocated from the intestinal lumen. Toll- like receptors, the bacterial recognition receptors, are expressed both on enteric nerves and smooth muscle and are emerging as potential mediators between microbiota and the enteric neuromuscular apparatus. Furthermore, the ongoing studies on probiotics support the hypothesis that the neuromuscular apparatus may represent a target of intervention, thus opening new physiopathological and therapeutic scenarios

The host metabolite D-serine contributes to bacterial niche specificity through gene selection

Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host–pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an ‘evolutionary incompatibility’ between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity

Biomarkers for monitoring intestinal health in poultry : present status and future perspectives

Intestinal health is determined by host (immunity, mucosal barrier), nutritional, microbial and environmental factors. Deficiencies in intestinal health are associated with shifts in the composition of the intestinal microbiome (dysbiosis), leakage of the mucosal barrier and/or inflammation. Since the ban on growth promoting antimicrobials in animal feed, these dysbiosis-related problems have become a major issue, especially in intensive animal farming. The economical and animal welfare consequences are considerable. Consequently, there is a need for continuous monitoring of the intestinal health status, particularly in intensively reared animals, where the intestinal function is often pushed to the limit. In the current review, the recent advances in the field of intestinal health biomarkers, both in human and veterinary medicine are discussed, trying to identify present and future markers of intestinal health in poultry. The most promising new biomarkers will be stable molecules ending up in the feces and litter that can be quantified, preferably using rapid and simple pen-side tests. It is unlikely, however, that a single biomarker will be sufficient to follow up all aspects of intestinal health. Combinations of multiple biomarkers and/or metabarcoding, metagenomic, metatranscriptomic, metaproteomic and metabolomic approaches will be the way to go in the future. Candidate biomarkers currently are being investigated by many research groups, but the validation will be a major challenge, due to the complexity of intestinal health in the field

Progress and problems in vaccination against necrotic enteritis in broiler chickens

Necrotic enteritis in broilers is caused by Clostridium perfringens type A strains that produce the NetB toxin. Necrotic enteritis is one of the gastrointestinal diseases in poultry that has gained worldwide importance during the last decade due to efforts to improve broiler performance. Prevention strategies include avoiding predisposing factors, such as coccidiosis, and in-feed supplementation with a variety of feed additives. However, vaccination with modified toxin or other secreted immunogenic proteins seems a logical preventive tool for protection against a toxin-producing bacterium. Formalin-inactivated crude supernatant has been used initially for vaccination. Several studies have been carried out recently to identify the most important immunogenic and protective proteins that can be used for vaccination. These include the NetB toxin, as well as a number of other proteins. There is evidence that immunization with single proteins is not protective against severe challenge and that combinations of different antigens are needed. Most published studies have used multiple dosage vaccination regimens that are not relevant for practical use in the broiler industry. Single vaccination regimens for 1-day-old chicks appear to be non-protective. This review describes the history of vaccination strategies against necrotic enteritis in broilers and gives an update on future vaccination strategies that are applicable in the field. These may include breeder hen vaccination, in ovo vaccination and live attenuated vectors to be used in feed or in drinking water

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism.

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2(-/-) genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility

Cereal non-starch polysaccharides in pig diets

This thesis is based on three different studies comprising the weaning and the growing period, aiming at monitoring the influence of cereal non-starch polysaccharides (NSP) and dietary enzyme supplementation on gastro-intestinal processes in pigs. The diets were based on cereals and cereal by-products, and composed to contain different amounts of total as well as soluble NSP. Results from these studies have shown that with increased NSP content, the reduction in digestibility of organic matter (OM) was twice as high in the small intestine than in the total tract, both in newly weaned piglets and growing pigs. An increased proportion of insoluble NSP decreased the digestibility of OM and fibre components in the small intestine of the newly weaned piglets and in the total tract of growing pigs. The gut environment, as described by content and proportions of organic acids (OA) and pH, as well as total microbial populations and coliform diversity, was altered by NSP content and solubility, whereas enzyme supplementation influenced the distribution of OA in the small intestine. PVTC-cannulation did not influence the coliform flora, and results obtained from PVTC-cannulated pigs were concluded to reflect true intestinal conditions. In conclusion, these results indicate that the dietary content of total and soluble NSP influence gastro-intestinal processes such as digestion site, gut environment and microbial populations in different ways in newly weaned piglets and in growing pigs. Therefore, NSP constitute an important tool with possibilities to influence gut health in pigs, and may therefore offer prospects to optimise the feed for pigs of different age