Investigation of the gut microbiome, bile acid composition and host immunoinflammatory response in a model of azoxymethane-induced colon cancer at discrete timepoints.

BACKGROUND: Distinct sets of microbes contribute to colorectal cancer (CRC) initiation and progression. Some occur due to the evolving intestinal environment but may not contribute to disease. In contrast, others may play an important role at particular times during the tumorigenic process. Here, we describe changes in the microbiota and host over the course of azoxymethane (AOM)-induced tumorigenesis. METHODS: Mice were administered AOM or PBS and were euthanised 8, 12, 24 and 48 weeks later. Samples were analysed using 16S rRNA gene sequencing, UPLC-MS and qRT-PCR. RESULTS: The microbiota and bile acid profile showed distinct changes at each timepoint. The inflammatory response became apparent at weeks 12 and 24. Moreover, significant correlations between individual taxa, cytokines and bile acids were detected. One co-abundance group (CAG) differed significantly between PBS- and AOM-treated mice at week 24. Correlation analysis also revealed significant associations between CAGs, bile acids and the bile acid transporter, ASBT. Aberrant crypt foci and adenomas were first detectable at weeks 24 and 48, respectively. CONCLUSION: The observed changes precede host hyperplastic transformation and may represent early therapeutic targets for the prevention or management of CRC at specific timepoints in the tumorigenic process.

Microbial Metabolites as Molecular Mediators of Host-Microbe Symbiosis in Colorectal Cancer.

The symbiosis between the gut microbiota and the host has been identified as an integral part of normal human physiology and physiological development. Research in germ-free or gnotobiotic animals has demonstrated the importance of this symbiosis in immune, vascular, hepatic, respiratory and metabolic systems. Disruption of the microbiota can also contribute to disease, and the microbiota has been implicated in numerous intestinal and extra-intestinal pathologies including colorectal cancer. Interactions between host and microbiota can occur either directly or indirectly, via microbial-derived metabolites. In this chapter, we focus on two major products of microbial metabolism, short-chain fatty acids and bile acids, and their role in colorectal cancer. Short-chain fatty acids are the products of microbial fermentation of complex carbohydrates and confer protection against cancer risk, while bile acids are compounds which are endogenous to the host, but undergo microbial modification in the large intestine leading to alterations in their bioactivity. Lastly, we discuss the ability of microbial modulation to mediate cancer risk and the potential to harness this ability as a prophylactic or therapeutic treatment in colorectal cancer.

Aphis glycines virus 1, a new bicistronic virus with two functional internal ribosome entry sites, is related to a group of unclassified viruses in the Picornavirales.

A novel picorna-like virus, provisionally named Aphis glycines virus 1 (ApGlV1) was discovered by high-throughput sequencing of soybean total RNAs and detected in suction trap-collected Aphis glycines. The ApGlV1 genome contains two large ORFs organized similar to those of dicipiviruses in the Picornaviridae where ORFs 1 and 2 encode structural and nonstructural proteins, respectively. Both ORFs are preceded by internal ribosome entry site (IRES) elements. The 5' IRES was more active in dual luciferase activity assays than the IRES in the intergenic region. The ApGlV1 genome was predicted to encode a serine protease instead of a cysteine protease and showed very low aa sequence identities to recognized members of the Picornavirales. In phylogenetic analyses based on capsid protein and RNA-dependent RNA polymerase sequences, ApGlV1 consistently clustered with a group of unclassified bicistronic picorna-like viruses discovered from arthropods and plants that may represent a novel family in the order Picornavirales.

Mycobacterium chimaera infection following cardiac surgery in the United Kingdom: clinical features and outcome of the first 30 cases.

OBJECTIVES: Mycobacterium chimaera infection following cardiac surgery, due to contaminated cardiopulmonary bypass heater-cooler units, has been reported worldwide. However, the spectrum of clinical disease remains poorly understood. To address this, we report the clinical and laboratory features, treatment and outcome of the first 30 UK cases. METHODS: Case note review was performed for cases identified retrospectively through outbreak investigations and prospectively through ongoing surveillance. Case definition was Mycobacterium chimaera detected in any clinical specimen, history of cardiothoracic surgery with cardiopulmonary bypass, and compatible clinical presentation. RESULTS: Thirty patients were identified (28 with prosthetic material) exhibiting a spectrum of disease including prosthetic valve endocarditis (14/30), sternal wound infection (2/30), aortic graft infection (4/30) and disseminated (non-cardiac) disease (10/30). Patients presented a median of 14 months post surgery (maximum 5 years) most commonly complaining of fever and weight loss. Investigations frequently revealed lymphopenia, thrombocytopenia, liver cholestasis and non-necrotizing granulomatous inflammation. Diagnostic sensitivity for a single mycobacterial blood culture was 68% but increased if multiple samples were sent. In all, 27 patients started macrolide-based combination treatment and 14 had further surgery. To date, 18 patients have died (60%) a median of 30 months (interquartile range 20-39 months) after initial surgery. Survival analysis identified younger age, mitral valve surgery, mechanical valve replacement, higher serum sodium concentration and lower C-reactive protein as factors associated with better survival. CONCLUSIONS: Mycobacterium chimaera infection following cardiac surgery is associated with a wide spectrum of disease. The diagnosis should be considered in all patients who develop an unexplained illness following cardiac surgery.

Molecular characterization of a new soybean-infecting member of the genus Nepovirus identified by high-throughput sequencing.

The complete nucleotide sequence of a new soybean-infecting member of the genus Nepovirus (provisionally named "soybean latent spherical virus" [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788 nt) were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3' nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus.

Differential expression of key regulators of Toll-like receptors in ulcerative colitis and Crohn's disease: a role for Tollip and peroxisome proliferator-activated receptor gamma?

The innate immune system is currently seen as the probable initiator of events which culminate in the development of inflammatory bowel disease (IBD) with Toll-like receptors (TLRs) known to be involved in this disease process. Many regulators of TLRs have been described, and dysregulation of these may also be important in the pathogenesis of IBD. The aim of this study was to perform a co-ordinated analysis of the expression levels of both key intestinal TLRs and their inhibitory proteins in the same IBD cohorts, both ulcerative colitis (UC) and Crohn's disease (CD), in order to evaluate the potential roles of these proteins in the pathogenesis of IBD. Of the six TLRs (TLRs 1, 2, 4, 5, 6 and 9) examined, only TLR-4 was increased significantly in IBD, specifically in active UC. In contrast, differential alterations in expression of TLR inhibitory proteins were observed. A20 and suppressor of cytokine signalling 1 (SOCS1) were increased only in active UC while interleukin-1 receptor-associated kinase 1 (IRAK-m) and B cell lymphoma 3 protein (Bcl-3) were increased in both active UC and CD. In contrast, expression of both peroxisome proliferator-activated receptor gamma (PPARγ) and Toll interacting protein (Tollip) was decreased in both active and inactive UC and CD and at both mRNA and protein levels. In addition, expression of both PPARγ and A20 expression was increased by stimulation of a colonic epithelial cell line Caco-2 with both TLR ligands and commensal bacterial strains. These data suggest that IBD may be associated with distinctive changes in TLR-4 and TLR inhibitory proteins, implying that alterations in these may contribute to the pathogenesis of IBD.

Prostaglandin E2 and the EP receptors in malignancy: possible therapeutic targets?

Elevated expression of COX-2 and increased levels of PGE2 are found in numerous cancers and are associated with tumour development and progression. Although epidemiological, clinical and preclinical studies have shown that the inhibition of PGE2 synthesis through the use of either non-steroidal anti-inflammatory drugs (NSAIDs) or specific COX-2 inhibitors (COXibs) has the potential to prevent and treat malignant disease, toxicities due to inhibition of COX-2 have limited their use. Thus, there is an urgent need for the development of strategies whereby COX-2 activity may be reduced without inducing any side effects. The biological effects of PGE2 are mediated by signalling through four distinct E-type prostanoid (EP) receptors - EP1 , EP2 , EP3 and EP4 . In recent years, extensive effort has gone into elucidating the function of PGE2 and the EP receptors in health and disease, with the goal of creating selective inhibitors as a means of therapy. In this review, we focus on PGE2 , and in particular on the role of the individual EP receptors and their signalling pathways in neoplastic disease. As knowledge concerning the role of the EP receptors in cancer grows, so does the potential for exploiting the EP receptors as therapeutic targets for the treatment of cancer and metastatic disease.

Selective influence of host microbiota on cAMP-mediated ion transport in mouse colon.

More microbes are resident in the distal colon than any other part of the body, and this microbiota has the capacity to influence enteric nerve development, excitability, and gastrointestinal function. Germ-free (GF) mice are a valuable tool in interrogating the communication between microbiota and host. Despite the intimate relationship which exists between the microbiota and the colonic mucosa-submucosa, there is a paucity of studies examining the influence of the microbiota on secretogogue-evoked responses. To this end, we investigated both epithelial and neural-evoked ion transport, and the response elicited by two commensal organisms, in colonic mucosa-submucosa preparations from GF mice in Ussing chambers. Baseline electrical parameters, short-circuit current and transepithelial resistance, were comparable between tissues from GF and conventional animals. Noteworthy, however, was a hyper-responsiveness of GF colon to forskolin stimulation. In contrast, the absence of the microbiota did not influence the tissue response to bethanechol. Moreover, responses to the sodium-channel activator, veratridine, and the TRPV1 receptor agonist, capsaicin were preserved in GF mice relative to conventional tissues. Similarly, the short-circuit current response to two well-characterized commensal organisms occurred independent of an interaction with the host microbiota. This is the first comprehensive characterization of secretomotor responses in GF colon.

Identification of novel double-stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence.

Virulence and double-stranded RNA (dsRNA) profiles of 44 isolates of Fusarium virguliforme were compared. When grouped according to dsRNA profiles, isolates with large dsRNAs were significantly (P≤0.05) less virulent than isolates without dsRNAs. High-throughput sequence analysis of total RNA prepared from cultures with large dsRNAs identified two novel RNA viruses with genome sequences of approximately 9.3 kbp, which were named Fusarium virguliforme dsRNA mycovirus 1 and Fusarium virguliforme dsRNA mycovirus 2. The new viruses were most closely related to a group of unclassified viruses that included viruses of F. graminearum and Phlebiopsis gigantea and are related to members of the family Totiviridae.

Role of soybean mosaic virus-encoded proteins in seed and aphid transmission in soybean.

Soybean mosaic virus (SMV) is seed and aphid transmitted and can cause significant reductions in yield and seed quality in soybean (Glycine max). The roles in seed and aphid transmission of selected SMV-encoded proteins were investigated by constructing mutants in and chimeric recombinants between SMV 413 (efficiently aphid and seed transmitted) and an isolate of SMV G2 (not aphid or seed transmitted). As previously reported, the DAG amino acid sequence motif near the amino terminus of the coat protein (CP) was the major determinant in differences in aphid transmissibility of the two SMV isolates, and helper component proteinase (HC-Pro) played a secondary role. Seed transmission of SMV was influenced by P1, HC-Pro, and CP. Replacement of the P1 coding region of SMV 413 with that of SMV G2 significantly enhanced seed transmissibility of SMV 413. Substitution in SMV 413 of the two amino acids that varied in the CPs of the two isolates with those from SMV G2, G to D in the DAG motif and Q to P near the carboxyl terminus, significantly reduced seed transmission. The Q-to-P substitution in SMV 413 also abolished virus-induced seed-coat mottling in plant introduction 68671. This is the first report associating P1, CP, and the DAG motif with seed transmission of a potyvirus and suggests that HC-Pro interactions with CP are important for multiple functions in the virus infection cycle.

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells.

Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E(2) (PGE(2)), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE(2) increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E(2)-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE(2) positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE(2).

Fas ligand mediates immune privilege and not inflammation in human colon cancer, irrespective of TGF-beta expression.

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-beta1 (TGF-beta1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-beta1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-beta1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8-33-fold, n=16, P<0.001), together with a two-fold increase in TIL apoptosis (range 1.6-2.5-fold, n=14, P<0.001), relative to FasL-negative nests within the same tumours. The overall level of neutrophils present in all tumours examined was low (mean 0.3%, n=16), with FasL expression by tumour nests associated with a mean two-fold decrease in neutrophils, irrespective of TGF-beta1 expression. Together, our results suggest that tumour-expressed FasL is inhibitory rather than stimulatory towards antitumour immune responses.

Teen empowerment movement to prevent tobacco use by North Carolina's youth.

Strong youth and adult leadership and exemplary policy and program interventions put a unique synergy to work in North Carolina. The state aspires to be a model for the nation in overcoming barriers related to tobacco use prevention by empowering its greatest resource--youth. New grant funding is building the foundation for youth empowerment programs, and increased and sustained funding is being sought to significantly expand these efforts. Youth speak with a fresh voice, bringing energy and conviction, as well as non-traditional ideas and strategies to the achievement of their goals. By changing public opinion and influencing the actions of leaders in the nation's leading tobacco state, the North Carolina Tobacco Prevention and Control Branch will develop leadership for tobacco use prevention that will serve the entire nation.

Fas ligand upregulation is an early event in colonic carcinogenesis.

BACKGROUND/AIMS: Fas ligand (FasL) is a mediator of apoptosis via the Fas receptor (Fas/CD95/APO-1). Normal colonic epithelium expresses Fas, and appears to be relatively sensitive to Fas mediated apoptosis. Colonic adenocarcinomas coexpress FasL and Fas without undergoing widespread apoptosis. This study investigates the expression of FasL in colonic carcinogenesis from the earliest stages of the adenoma-carcinoma sequence. METHODS: FasL expression was determined in colonic adenomas (n = 38) of varying degrees of dysplasia and histological type by immunohistochemistry. Adenomas that contained areas of carcinomatous change were included (n = 12 of 38). Normal colonic epithelium (n = 10), hyperplastic polyps (n = 8), and serrated adenomas (n = 3) from patients without colonic adenocarcinomas were used for comparison. Cell death was detected in situ in adenomas using TUNEL (terminal transferase mediated dUTP nick end labelling). RESULTS: In normal colonic epithelium and hyperplastic polyps, FasL expression was restricted to the luminal surface of the crypts, where Fas-FasL coexpression was coincident with a high frequency of TUNEL positive epithelial cells. All adenomas (n = 38) had an altered distribution of positive FasL staining; FasL expression was found in most cells (> 70% of neoplastic cells). Expression of Fas was also detected throughout the adenomas, but coexpression of FasL and Fas was not associated with TUNEL positivity in most cells. CONCLUSIONS: FasL upregulation occurs early in the adenoma-carcinoma sequence of colon carcinogenesis, and is evident at the level of mild dysplasia. The lack of pronounced apoptosis in areas of adenomas coexpressing Fas and FasL suggests that colonocytes acquire resistance to Fas mediated apoptosis early in the transformation process.

Immune privilege or inflammation? Insights into the Fas ligand enigma.

Fas ligand (FasL) has become an enigmatic molecule: some evidence indicates that it contributes to immune privilege in tissues and tumors, whereas other data demonstrates that FasL can elicit inflammation. New findings may begin to reconcile the paradoxical effects of FasL.

Interventional treatment of lateral tunnel dehiscence in a total cavopulmonary connection using a balloon expandable covered stent.

In this paper we present a patient with dehiscence of an intra-atrial tunnel previously constructed during a total cavopulmonary connection procedure. We describe the use of a custom made covered stent to seal off the dehisced segment, and abolish the intra-cardiac shunting. We believe this is the first account of such a procedure being undertaken.

Altered mechanisms of apoptosis in colon cancer: Fas resistance and counterattack in the tumor-immune conflict.

Fas (CD95/APO-1) is a cell surface "death receptor" that mediates apoptosis upon engagement by its ligand, FasL. Fas-mediated apoptosis of lymphocytes normally serves immunoregulatory roles, including tolerance acquisition, immune response termination, and maintenance of immune privilege in certain organs. Colon tumors can exploit this lymphocyte death program by expressing FasL. This may enable colon tumors to mount a "Fas counterattack" against antitumor lymphocytes, impairing antitumor immune responses. FasL-expressing colon tumor-derived cell lines can trigger Fas-mediated apoptosis of cocultured T cells in vitro. FasL expressed in esophageal cancer has been significantly associated with apoptosis and depletion of tumor-infiltrating lymphocytes (TIL) in vivo. FasL may also facilitate metastatic colonization of Fas-sensitive organs such as the liver, by inducing apoptosis of target organ cells. Normal colonic epithelial cells express Fas and are relatively sensitive to Fas-mediated apoptosis. By contrast, colon tumor-derived cell lines are usually resistant to induction of Fas-mediated apoptosis, and colon cancer cells frequently coexpress Fas and FasL. The mechanisms allowing resistance to Fas-mediated apoptosis are complex, and defects have been identified at several levels of Fas signal transduction. The "Bcl-2 rheostat" may be pitched against apoptosis in colon cancer, inasmuch as overexpression of Bcl-2, downregulation of Bak, and mutation of Bax are common defects in colon tumors. Caspase-1 is also downregulated in colon cancer. The high frequency of p53 mutations in late-stage cancers may also inhibit Fas signaling. Fundamental defects in apoptosis signaling may contribute to both immuno- and chemoresistance in colon cancer and allow expression of FasL to counterattack antitumor lymphocytes.

International surveillance of blood stream infections due to Candida species in the European SENTRY Program: species distribution and antifungal susceptibility including the investigational triazole and echinocandin agents. SENTRY Participant Group (Europe).

The SENTRY Antimicrobial Surveillance Program, an international study of blood stream infections (BSIs), detected 170 episodes of candidemia in 20 European medical centers (13 nations) between January and December, 1997. Twenty-three percent of the candidal BSI occurred in patients hospitalized in an intensive care unit, 21% in patients in an internal medicine service, 13% in patients in a surgical service, and 9% in patients in an oncology service. Overall, 53% of the BSI were attributable to Candida albicans followed in prevalence by C. parapsilosis (21%), C. glabrata (12%), C. tropicalis (6%), C. famata (2%), C. krusei (1%), and C. inconspicua (1%). As observed previously in Canada and Latin America, C. parapsilosis and not C. glabrata, was the most common non-albicans species causing yeast BSI in Europe. The proportion of these candidemias attributable to C. albicans varied widely from 0-100% among the 20 European centers. Among the different species of Candida, resistance to fluconazole (MIC, > or = 64 micrograms/mL) and itraconazole (MIC, > or = 1.0 microgram/mL) was observed with C. glabrata and C. krusei and was observed more rarely among other species (e.g., C. inconspicua). Isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. guilliermondii were all highly susceptible to both fluconazole and itraconazole. Furthermore, the investigational triazoles (BMS-207147, Sch 56592, and voriconazole) and an echinocandin (MK-0991) all demonstrated potent in vitro activity (MIC90s, 0.5, 0.5, 1.0, and 2.0 micrograms/mL, respectively) against these isolates. Continued surveillance at an international level will be important to monitor trends in species distribution and antifungal susceptibility among invasive strains of Candida.

Outcomes of transposition of the great arteries in the ear of atrial inflow correction.

OBJECTIVE: To examine long term morbidity and mortality following atrial inflow corrective procedures for transposition of the great arteries (TGA) and to investigate factors that influence morbidity and mortality. DESIGN: Retrospective cohort study from a single centre. SETTING: Cardiology and cardiothoracic surgical unit in a large tertiary referral centre. PATIENTS: All 130 patients who had TGA diagnosed between August 1972 and May 1988 and were considered suitable for atrial inflow correction; 109 of these underwent surgery (operative cohort: 84 Mustard operations and 25 Senning operations); 95 survived to hospital discharge (hospital surviving cohort). MAIN OUTCOME MEASURES: Death and cardiac events. RESULTS: There were relatively good long term results from atrial inflow correction for TGA with 5, 10, and 15 year survivals of 77.3%, 75.9%, and 71.3%. However, there was an appreciable incidence of late cardiac death and events, with 5, 10, and 15 year cardiac event-free survivals of 74.5%, 67.1%, and 39.6%. Supraventricular tachycardia was the only significant risk factor for late cardiac death (relative risk 8.72, 95% confidence interval, 2.86 to 26.64). Senning patients had better event-free survival (p = 0.04). CONCLUSIONS: Atrial inflow correction for TGA has a reasonably good 15 year survival (71.3%), but there is an appreciable incidence of late cardiac deaths and events (15 year event-free survival 39.6%). The Senning procedure is preferable to the Mustard procedure for cases unsuitable for arterial switching.

Recurrence risks in offspring of adults with major heart defects: results from first cohort of British collaborative study.

BACKGROUND: Congenital heart defects are generally assumed to have a multifactorial aetiology. We have tested this hypothesis by studying adults with heart defects and their families. METHODS: We identified 1094 patients who survived surgery for major cardiac defects before 1970. We chose individuals with disturbance of situs or segmental connection, with atrioventricular septal defect or with tetralogy of Fallot. After exclusion and non-participation, 727 individuals were traced. Each was visited by an investigator and completed a detailed questionnaire. If possible, all "normal" offspring were examined by a paediatric cardiologist. FINDINGS: The 727 individuals had 393 live offspring. There were 71 miscarriages and five terminated pregnancies. Overall, we found recurrent heart defects in 16 liveborn offspring--a recurrence risk of 4.1%. This result differed significantly from sibling risk (2.1%; p=0.021). More congenital heart defects occurred in the offspring of affected women than in those of affected men (p=0.047); when all malformations (cardiac and non-cardiac) in the offspring were taken into account the excess was more significant (p=0.032). We found an excess of miscarriages in the offspring of affected women (p=0.001). In tetralogy of Fallot, heart defects occurred in seven (3.1%) of 223 offspring, 12 (2.2%) of 539 siblings, five (0.3%) of 1575 second-degree relatives, and eight (0.3%) of 2728 third-degree relatives. INTERPRETATION: Our findings do not support a polygenic basis for all heart defects. Atrioventricular septal defect seems to be a single-gene defect and tetralogy of Fallot a polygenic disorder with a small number of interacting genes. Our data suggest that isolated transposition of the great arteries is a sporadic defect.