Chemistry meets biology in colitis-associated carcinogenesis

The intestine comprises an exceptional venue for a dynamic and complex interplay of numerous chemical and biological processes. Here, multiple chemical and biological systems, including the intestinal tissue itself, its associated immune system, the gut microbiota, xenobiotics, and metabolites meet and interact to form a sophisticated and tightly regulated state of tissue homoeostasis. Disturbance of this homeostasis can cause inflammatory bowel disease (IBD)—a chronic disease of multifactorial etiology that is strongly associated with increased risk for cancer development. This review addresses recent developments in research into chemical and biological mechanisms underlying the etiology of inflammation-induced colon cancer. Beginning with a general overview of reactive chemical species generated during colonic inflammation, the mechanistic interplay between chemical and biological mediators of inflammation, the role of genetic toxicology, and microbial pathogenesis in disease development are discussed. When possible, we systematically compare evidence from studies utilizing human IBD patients with experimental investigations in mice. The comparison reveals that many strong pathological and mechanistic correlates exist between mouse models of colitis-associated cancer, and the clinically relevant situation in humans. We also summarize several emerging issues in the field, such as the carcinogenic potential of novel inflammation-related DNA adducts and genotoxic microbial factors, the systemic dimension of inflammation-induced genotoxicity, and the complex role of genome maintenance mechanisms during these processes. Taken together, current evidence points to the induction of genetic and epigenetic alterations by chemical and biological inflammatory stimuli ultimately leading to cancer formation.Massachusetts Institute of Technology. Center for Environmental Health Sciences (ES002109)National Institutes of Health (U.S.) (NIH (CA26731)

An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants

The oxidation of guanine to 8-oxo-2′-deoxyguanosine (8-oxo-dG) is one of the most abundant and best studied oxidative DNA lesions and is commonly used as a biomarker for oxidative stress. Over the last decades, various methods for the detection of DNA oxidation products have been established and optimized. However, some of them lack sensitivity or are prone to artifact formation, while others are time-consuming, which hampers their application in screening approaches. In this study, we present a formamidopyrimidine glycosylase (Fpg)-based method to detect oxidative lesions in isolated DNA using a modified protocol of the automated version of the fluorimetric detection of alkaline DNA unwinding (FADU) method, initially developed for the measurement of DNA strand breaks (Moreno-Villanueva et al., 2009. BMC Biotechnol. 9, 39). The FADU-Fpg method was validated using a plasmid DNA model, mimicking mitochondrial DNA, and the results were correlated to 8-oxo-dG levels as measured by LC–MS/MS. The FADU-Fpg method can be applied to analyze the potential of compounds to induce DNA strand breaks and oxidative lesions, as exemplified here by treating plasmid DNA with the peroxynitrite-generating molecule Sin-1. Moreover, this method can be used to screen DNA-protective effects of antioxidant substances, as exemplified here for a small-molecule, i.e., uric acid, and a protein, i.e., manganese superoxide dismutase, both of which displayed a dose-dependent protection against the generation of oxidative DNA lesions. In conclusion, the automated FADU-Fpg method offers a rapid and reliable measurement for the detection of peroxynitrite-mediated DNA damage in a cell-free system, rendering it an ideal method for screening the DNA-protective effects of antioxidant compounds.Deutsche Forschungsgemeinschaft (Grant BU 698/6-1)National Institutes of Health (U.S.) (Grant ES002109)National Institutes of Health (U.S.) (Grant CA026731

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2[superscript −/−] mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.National Institutes of Health (U.S.) (Grant CA26731)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant ES002109))Massachusetts Institute of Technology (Merck Fellowship)German Academic Exchange Service (Fellowship

Helicobacter cinaedi Induced Typhlocolitis in Rag-2-Deficient Mice

Background Helicobacter cinaedi, an enterohepatic helicobacter species (EHS), is an important human pathogen and is associated with a wide range of diseases, especially in immunocompromised patients. It has been convincingly demonstrated that innate immune response to certain pathogenic enteric bacteria is sufficient to initiate colitis and colon carcinogenesis in recombinase-activating gene (Rag)-2-deficient mice model. To better understand the mechanisms of human IBD and its association with development of colon cancer, we investigated whether H. cinaedi could induce pathological changes noted with murine enterohepatic helicobacter infections in the Rag2[superscript −/−] mouse model. Materials and Methods Sixty 129SvEv Rag2[superscript −/−] mice mouse were experimentally or sham infected orally with H. cinaedi strain CCUG 18818. Gastrointestinal pathology and immune responses in infected and control mice were analyzed at 3, 6 and 9 months postinfection (MPI). H. cinaedi colonized the cecum, colon, and stomach in infected mice. Results H. cinaedi induced typhlocolitis in Rag2[superscript −/−] mice by 3 MPI and intestinal lesions became more severe by 9 MPI. H. cinaedi was also associated with the elevation of proinflammatory cytokines, interferon-γ, tumor-necrosis factor-α, IL-1β, IL-10; iNOS mRNA levels were also upregulated in the cecum of infected mice. However, changes in IL-4, IL-6, Cox-2, and c-myc mRNA expressions were not detected. Conclusions Our results indicated that the Rag2[superscript −/−] mouse model will be useful to continue investigating the pathogenicity of H. cinaedi, and to study the association of host immune responses in IBD caused by EHS.United States. National Institutes of Health (R01-0D011141)United States. National Institutes of Health (R01-CA067529)United States. National Institutes of Health (P30-ES002109)United States. National Institutes of Health (P01-CA026731

Perforation of the lower topsoil and the upper subsoil in a beet-cereal crop rotation after 24 years of tillage with and without plowing with and without green manure

When plowing, there is a risk that over time a compacted, poorly perforated plow pan will form directly under the plowing horizon (crumb base) at a depth of 30 to 35 cm. However, shallow tillage is not without its problems either. The weight of heavy machinery, especially if the soil is too moist, can compact the deeper layer of topsoil at a depth of 20 to 30 cm (deep topsoil) over time. Using double-ring infiltrometers, the perforation of the deep topsoil and the crumb base was investigated in a long-term tillage experiment with and without green manure. In the long term, plowing was by no means detrimental to soil perforation. However, regular intercropping with yellow mustard was beneficial in the long term. In regenerative agriculture, particular attention should therefore be paid to the cultivation of catch crops with allorhizal root systems (instead of plowless tillage), as these significantly improve soil perforation

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10[superscript −/−] mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10[superscript −/−] mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.National Institutes of Health (U.S.) (Grant NIH P01-CA26731)National Institutes of Health (U.S.) (Grant NIH P30ES0026731)National Institutes of Health (U.S.) (Grant NIH R01-OD011141

Earthworms in the soil under a beet-cereal rotation after 24 years of no plowing with and without green manure

Annual plowing is helpful in controlling weeds, but it can also be detrimental to earthworms in the soil. In a now 24-year long-term trial in the dry¬lands of southwest Germany, it was investigated how the intensity of tillage (plow 30 cm deep vs. goose share culti¬vator 15 cm deep) and the implementation of a green manure every 3rd year within the crop rotation (with vs. without) affects earthworm population. The follo¬wing two questions were the main focus: (1) Does the earthworm population suffer over time due to the low humus regene¬ration capacity of the beet-cereal crop rota¬tion with straw removal and without organic fertilization? (2) Can the negative effect of low humus-regeneration capacity be compensated by earthworm-promoting measures such as no plowing and green manuring? In the 9 years from the first to the second campaign, earthworm biomass decreased by about 30 % (mean across all variants). With one exception, earthworm biomass was always lower in the plowed soil than in the corresponding cultivator variant. For endogeic earthworms, plowing - especially in combination with green manure - was even rather positive. Generally, earthworms benefited more from green manuring than from reduced tillage

Perforation of the lower topsoil and the upper subsoil in a beet-cereal crop rotation after 24 years of tillage with and without plowing with and without green manure

When plowing, there is a risk that over time a compacted, poorly perforated plow pan will form directly under the plowing horizon (crumb base) at a depth of 30 to 35 cm. However, shallow tillage is not without its problems either. The weight of heavy machinery, especially if the soil is too moist, can compact the deeper layer of topsoil at a depth of 20 to 30 cm (deep topsoil) over time. Using double-ring infiltrometers, the perforation of the deep topsoil and the crumb base was investigated in a long-term tillage experiment with and without green manure. In the long term, plowing was by no means detrimental to soil perforation. However, regular intercropping with yellow mustard was beneficial in the long term. In regenerative agriculture, particular attention should therefore be paid to the cultivation of catch crops with allorhizal root systems (instead of plowless tillage), as these significantly improve soil perforation

Serum Metabolome and Lipidome Changes in Adult Patients with Primary Dengue Infection

10.1371/journal.pntd.0002373PLoS Neglected Tropical Diseases78

Restriction of AID activity and somatic hypermutation by PARP-1

Affinity maturation of the humoral immune response depends on somatic hypermutation (SHM) of immunoglobulin (Ig) genes, which is initiated by targeted lesion introduction by activation-induced deaminase (AID), followed by error-prone DNA repair. Stringent regulation of this process is essential to prevent genetic instability, but no negative feedback control has been identified to date. Here we show that poly(ADP-ribose) polymerase-1 (PARP-1) is a key factor restricting AID activity during somatic hypermutation. Poly(ADP-ribose) (PAR) chains formed at DNA breaks trigger AID-PAR association, thus preventing excessive DNA damage induction at sites of AID action. Accordingly, AID activity and somatic hypermutation at the Ig variable region is decreased by PARP-1 activity. In addition, PARP-1 regulates DNA lesion processing by affecting strand biased A:T mutagenesis. Our study establishes a novel function of the ancestral genome maintenance factor PARP-1 as a critical local feedback regulator of both AID activity and DNA repair during Ig gene diversification