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PDF file - 110K, Genes differentially expressed by treatment. A. 169 downregulated and 194 upregulated genes in infected E2 male mice compared to infected untreated male mice. B. 69 downregulated and 75 upregulated genes in infected TAM male mice compared to infected untreated male mice. C. 24 downregulated and 37 upregulated genes in both infected E2 and TAM male mice compared to infected untreated male mice.

PDF file - 48K, Biological functions and canonical pathways associated with both E2 and TAM treatment of infected males and genes associated with function or pathway. A. Diseases and disorders. B. Molecular and Cellular functions. C. Physiological System and Development and Functions. D. Canonical pathways.

Helicobacter pylori infection promotes male predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies showed that prophylactic 17β-estradiol (E2) in INS-GAS mice decreases H. pylori–induced carcinogenesis. We examined the effect of E2 and tamoxifen (TAM) on H. pylori–induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks postinfection (WPI), mice were implanted with E2, TAM, both E2 and TAM, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression, and immune cell infiltration were evaluated and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori–infected males receiving E2 and/or TAM, whereas 40% of infected untreated males developed gastric cancer. E2, TAM, and their combination significantly reduced gastric precancerous lesions in infected males compared with infected untreated males (P < 0.001, 0.01, and 0.01, respectively). However, TAM did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or TAM (n = 363 and n = 144, Q < 0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or TAM deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared with controls, E2 decreased gastric mRNA (Q < 0.05) and serum levels (P < 0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P < 0.01). Prevention of H. pylori–induced gastric cancer by E2 and TAM may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts, and downregulation of oncogenic pathways. Cancer Prev Res; 4(9); 1426–35. ©2011 AACR.

PDF file - 45K, Individual histological parameters in the corpus and antrum at 16 and 28 weeks of H. pylori infection. A) Corpus pathology at 16 WPI. B) Corpus pathology at 28 WPI. C) Antrum pathology at 16 WPI. D) Antrum pathology at 28 WPI.

PDF file - 49K, Biological functions and canonical pathways associated with TAM treatment of infected males and genes associated with function or pathway. A. Diseases and disorders. B. Molecular and Cellular functions. C. Physiological System and Development and Functions. D. Canonical pathways.

PDF file - 529K, Significant molecular networks affected by the common genes between E2 and TAM treatments (P=10-45). P-values represent the probability of these interactions occurring by chance.

PDF file - 52K, Networks associated to differentially expressed genes by Ingenuity Pathway Analysis. Genes in capital letters represent differentially expressed genes found within the network. A. Top networks associated with E2 treatment of infected male mice. B. Top networks associated with TAM treatment of infected male mice. C. Top 3 networks associated with commonly induced genes in both E2 and TAM treatments.

PDF file - 915K, Significant molecular networks affected by E2 treatment (P=10-50). P-values represent the probability of these interactions occurring by chance.

PDF file - 419K, Significant molecular networks affected by Tamoxifen treatment (P=10-45). P-values represent the probability of these interactions occurring by chance.

PDF file - 53K, Biological functions and canonical pathways associated with E2 treatment of infected males and genes associated with function or pathway. A. Diseases and disorders. B. Molecular and Cellular functions. C. Physiological System and Development and Functions. D. Canonical pathways.

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.

Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets. To understand the role of the microbiome in GI diseases, we characterized the gut microbiome of 91 healthy marmosets (303 samples) and 59 marmosets diagnosed with inflammatory bowel disease (IBD) (200 samples). Healthy marmosets exhibited “humanized,” Bacteroidetes-dominant microbiomes. After up to 2 years of standardized diet, housing and husbandry, marmoset microbiomes could be classified into four distinct marmoset sources based on Prevotella and Bacteroides levels. Using a random forest (RF) model, marmosets were classified by source with an accuracy of 93% with 100% sensitivity and 95% specificity using abundance data from 4 Prevotellaceae amplicon sequence variants (ASVs), as well as single ASVs from Coprobacter, Parabacteroides, Paraprevotella, Phascolarctobacterium, Oribacterium and Fusobacterium. A single dysbiotic IBD state was not found across all marmoset sources, but IBD was associated with lower alpha diversity and a lower Bacteroides:Prevotella copri ratio within each source. IBD was highest in a Prevotella-dominant cohort, and consistent with Prevotella-linked diseases, pro-inflammatory genes in the jejunum were upregulated. RF analysis of serum biomarkers identified serum calcium, hemoglobin and red blood cell (RBC) counts as potential biomarkers for marmoset IBD. This study characterizes the microbiome of healthy captive common marmosets and demonstrates that source-specific microbiomes can be retained despite standardized diets and husbandry practices. Marmosets with IBD had decreased alpha diversity and a shift in the ratio of Bacteroides:Prevotella copri compared to healthy marmosets.

Current methods for detecting mites in mouse colonies have limitations in terms of cost, accuracy, and throughput. To address these limitations, we developed PCR assays to detect Myocoptes musculinus in fecal samples. Using a newly generated ribosomal RNA sequence of M. musculinus (MC28S), we developed PCR and qPCR assays capable of detecting M. musculinus mites or eggs ingested during grooming. To determine our ability to detect mites, we tested fur swabs and feces from mouse colonies experimentally infested with M. musculinus and Demodex musculi, 2) Myobia musculi and Radfordia affinis, 3) M. musculinus and M. musculi, and 4) no mites (negative control). The MC28S PCR and qPCR assays positively identified M. musculinus in groups 1 and 3. The MC28S PCR assay detected M. musculinus in 9 of 10 fecal samples from known-positive animals, whereas the qPCR assay correctly identified M. musculinus in all 10 fecal samples. To our knowledge, this report is the first description of PCR-based detection of murine mites in feces. By eliminating the need for pelt examinations, mite detection from fecal samples can facilitate mite detection in sentinel or quarantine programs.

Cyclomodulins are virulence factors that modulate cellular differentiation, apoptosis, and proliferation. These include colibactin (pks), cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt). Pathogenic pks+, cnf+, and cdt+ E. coli strains are associated with inflammatory bowel disease (IBD) and colorectal cancer in humans and animals. Captive marmosets are frequently afflicted with IBD-like disease, and its association with cyclomodulins is unknown. Cyclomodulin-encoding E. coli rectal isolates were characterized using PCR-based assays in healthy and clinically affected marmosets originating from three different captive sources. 139 E. coli isolates were cultured from 122 of 143 marmosets. The pks gene was detected in 56 isolates (40%), cnf in 47 isolates (34%), and cdt in 1 isolate (0.7%). The prevalences of pks+ and cnf+ E. coli isolates were significantly different between the three marmoset colonies. 98% of cyclomodulin-positive E. coli belonged to phylogenetic group B2. Representative isolates demonstrated cyclomodulin cytotoxicity, and serotyping and whole genome sequencing were consistent with pathogenic E. coli strains. However, the presence of pks+, cnf+, or cdt+ E. coli did not correlate with clinical gastrointestinal disease in marmosets. Cyclomodulin-encoding E. coli colonize laboratory common marmosets in a manner dependent on the source, potentially impacting reproducibility in marmoset models.

Chronic gastrointestinal (GI) diseases are the most common diseases in captive marmosets. The gut microbiome of healthy (n=91), inflammatory bowel disease (IBD) (n=59), and duodenal ulcer/stricture (n=23) captive marmosets was characterized. Healthy marmosets exhibited a “humanized,” Bacteroidetes-dominant microbiome. Despite standardized conditions, cohorts subdivided into Prevotella- and Bacteroides-dominant groups based on marmoset source. IBD was highest in a Prevotella-dominant cohort while strictures were highest in a Bacteroides-dominant cohort. Stricture-associated dysbiosis was characterized by Anaerobiospirillum loss and Clostridium perfringens increases. Stricture tissue presented upregulation of lipid metabolism genes and increased abundance of C. perfringens, a causative agent of GI diseases and intestinal strictures in humans. IBD was associated with a lower Bacteroides:P. copri ratio within each source. Consistent with Prevotella-linked diseases, pro-inflammatory genes were upregulated. This report highlights the humanization of the captive marmoset microbiome and its potential as a “humanized” animal model of C. perfringens-induced enteritis/strictures and P. copri-associated IBD.

A fast-growing Mycobacterium species was cultured from draining, purulent lesions on the caudal abdomen of a 12-year-old male domestic long-haired cat. Whole-genome sequencing identified the organism as Mycobacterium porcinum.

© 2020, Society for Mucosal Immunology. Mice deficient in the IL-10 pathway are the most widely used models of intestinal immunopathology. IL-17A is strongly implicated in gut disease in mice and humans, but conflicting evidence has drawn IL-17’s role in the gut into question. IL-22 regulates antimicrobial and repair activities of intestinal epithelial cells (IECs) and is closely associated with IL-17A responses but it’s role in chronic disease is uncertain. We report that IL-22, like IL-17A, is aberrantly expressed in colitic Il10−/− mice. While IL-22+Th17 cells were elevated in the colon, IL-22-producing ILC3s were highly enriched in the small intestines of Il10−/− mice. Remarkably, Il10−/−Il22−/− mice did not develop colitis despite retaining high levels of Th17 cells and remaining colonized with colitogenic Helicobacter spp. Accordant with IL-22-induced IEC proliferation, the epithelia hyperplasia observed in Il10−/− animals was reversed in Il10−/−Il22−/− mice. Also, the high levels of antimicrobial IL-22-target genes, including Reg3g, were normalized in Il10−/−Il22−/− mice. Consistent with a heightened antimicrobial environment, Il10−/− mice had reduced diversity of the fecal microbiome that was reestablished in Il10−/−Il22−/− animals. These data suggest that spontaneous colitis in Il10−/− mice is driven by IL-22 and implicates an underappreciated IL-10/IL-22 axis in regulating intestinal homeostasis.

© 2020, © 2020 Taylor & Francis Group, LLC. Infection with Helicobacter pylori causes chronic inflammation and is a risk factor for gastric cancer. Antibiotic treatment or increased dietary folate prevents gastric carcinogenesis in male INS-GAS mice. To determine potential synergistic effects, H. pylori-infected male INS-GAS mice were fed an amino acid defined (AAD) diet with increased folate and were treated with antibiotics after 18 weeks of H. pylori infection. Antibiotic therapy decreased gastric pathology, but dietary folate had no effect. However, the combination of antibiotics and the AAD diet induced anemia, gastric hemorrhage, and mortality. Clinical presentation suggested hypovitaminosis K potentially caused by dietary deficiency and dysbiosis. Based on current dietary guidelines, the AAD diet was deficient in vitamin K. Phylloquinone administered subcutaneously and via a reformulated diet led to clinical improvement with no subsequent mortalities and increased hepatic vitamin K levels. We characterized the microbiome and menaquinone profiles of antibiotic-treated and antibiotic-free mice. Antibiotic treatment decreased the abundance of menaquinone producers within orders Bacteroidales and Verrucomicrobiales. PICRUSt predicted decreases in canonical menaquinone biosynthesis genes, menA and menD. Reduction of menA from Akkermansia muciniphila, Bacteroides uniformis, and Muribaculum intestinale were confirmed in antibiotic-treated mice. The fecal menaquinone profile of antibiotic-treated mice had reduced MK5 and MK6 and increased MK7 and MK11 compared to antibiotic-free mice. Loss of menaquinone-producing microbes due to antibiotics altered the enteric production of vitamin K. This study highlights the role of diet and the microbiome in maintaining vitamin K homeostasis.

Current methods for detecting mites in mouse colonies have limitations in terms of cost, accuracy, and throughput. To address these limitations, we developed PCR assays to detect Myocoptes musculinus in fecal samples. Using a newly generated ribosomal RNA sequence of M. musculinus (MC28S), we developed PCR and qPCR assays capable of detecting M. musculinus mites or eggs ingested during grooming. To determine our ability to detect mites, we tested fur swabs and feces from mouse colonies experimentally infested with M. musculinus and Demodex musculi, 2) Myobia musculi and Radfordia affinis, 3) M. musculinus and M. musculi, and 4) no mites (negative control). The MC28S PCR and qPCR assays positively identified M. musculinus in groups 1 and 3. The MC28S PCR assay detected M. musculinus in 9 of 10 fecal samples from known-positive animals, whereas the qPCR assay correctly identified M. musculinus in all 10 fecal samples. To our knowledge, this report is the first description of PCR-based detection of murine mites in feces. By eliminating the need for pelt examinations, mite detection from fecal samples can facilitate mite detection in sentinel or quarantine programs.

Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2−/-Il10−/− gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice., National Institutes of Health (U.S.) (Grant R01–0D01141), National Institutes of Health (U.S.) (Grant T32–0D010978), National Institutes of Health (U.S.) (Grant P30-ES002109), National Institutes of Health (U.S.) (Grant P01-CA26731), National Institutes of Health (U.S.) (Grant R01-CA080024)

The common marmoset (Callithrix jacchus) is increasingly used as an animal model for biomedical research; however, gastrointestinal diseases causing significant morbidity are endemic in many captive marmoset colonies. Establishing gut microbiome patterns in a marmoset colony may aid in clinical decision-making and model reproducibility. A standardized method of sample collection and storage is essential for proper interpretation of microbiome data. While microbiome studies commonly utilize fecal samples, the goal of this study was to determine whether the microbiome profile from a rectal swab performed on a sedated animal was comparable to the microbiome profile from a fecal sample. During routine physical exams, paired fecal and rectal swab samples were collected from each of 23 marmosets. DNA was extracted from all fecal and rectal swab samples and 16S ribosomal RNA gene sequences were amplified and analyzed. Initial comparison of the relative abundance of bacterial phyla between paired samples had a r2 value of 0.70 with S of 0.08 with no significant differences in α and β diversity metrics between fecal and rectal samples. Initial analysis however, revealed 5 discordant fecal-rectal pairs which corresponded only with the 5 rectal swabs that were classified as free of visible fecal matter during collection. Exclusion of these 5 pairs resulted in an optimized fit of the data as evidenced by a r2 value of 0.91 with S of 0.05. These results demonstrate that rectal swabs are a reliable method for profiling the fecal microbiome in the marmoset since the bacterial composition from a rectal swab with visible fecal contents correlated well with the bacterial composition from a fecal sample from the same marmoset. This study highlights the importance of standardized sample collection methods and exclusion of inappropriate samples.

A urease-negative, fusiform, novel bacterium named Helicobacter saguini was isolated from the intestines and feces of cotton-top tamarins (CTTs) with chronic colitis. Helicobacter sp. was detected in 69% of feces or intestinal samples from 116 CTTs. The draft genome sequence, obtained by Illumina MiSeq sequencing, for H. saguini isolate MIT 97-6194-5, consisting of ∼2.9 Mb with a G+C content of 35% and 2,704 genes, was annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline. H. saguini contains homologous genes of known virulence factors found in other enterohepatic helicobacter species (EHS) and H. pylori . These include flagellin, γ-glutamyl transpeptidase ( ggt ), collagenase, the secreted serine protease htrA , and components of a type VI secretion system, but the genome does not harbor genes for cytolethal distending toxin ( cdt ). H. saguini MIT 97-6194-5 induced significant levels of interleukin-8 (IL-8) in HT-29 cell culture supernatants by 4 h, which increased through 24 h. mRNAs for the proinflammatory cytokines IL-1β, tumor necrosis factor alpha (TNF-α), IL-10, and IL-6 and the chemokine CXCL1 were upregulated in cocultured HT-29 cells at 4 h compared to levels in control cells. At 3 months postinfection, all H. saguini -monoassociated gnotobiotic C57BL/129 IL-10 −/− mice were colonized and had seroconverted to H. saguini antigen with a significant Th1-associated increase in IgG2c ( P < 0.0001). H. saguini induced a significant typhlocolitis, associated epithelial defects, mucosa-associated lymphoid tissue (MALT) hyperplasia, and dysplasia. Inflammatory cytokines IL-22, IL-17a, IL-1β, gamma interferon (IFN-γ), and TNF-α, as well as inducible nitric oxide synthase (iNOS) were significantly upregulated in the cecal tissues of infected mice. The expression of the DNA damage response molecule γ-H2AX was significantly higher in the ceca of H. saguini -infected gnotobiotic mice than in the controls. This model using a nonhuman primate Helicobacter sp. can be used to study the pathogenic potential of EHS isolated from primates with naturally occurring inflammatory bowel disease (IBD) and colon cancer.

C57BL/6 (B6) mice from Taconic Sciences (Tac) and the Jackson Laboratory (Jax) were infected with H. pylori PMSS1 (Hp) for 16 week; there was no significant difference in the gastric histologic activity index between Hp infected Tac and Jax B6. However, the degree of gastric mucous metaplasia and Th1-associated IgG2c levels in response to Hp infection were increased in Tac mice over Jax mice, whereas the colonization levels of gastric Hp were higher by 8-fold in Jax B6 compared with Tac B6. Additionally, mRNA expression of gastric Il-1β, Il-17A and RegIIIγ were significantly lower in the infected Tac compared to the infected Jax mice. There were significant differences in the microbial community structures in stomach, colon, and feces between Jax and Tac B6 females. Differences in gastric microbial communities between Jax and Tac B6 females are predicted to affect the metagenome. Moreover, Hp infection perturbed the microbial community structures in the stomach, colon and feces of Jax mice, but only altered the colonic microbial composition of Tac mice. Our data indicate that the GI microbiome of Tac B6 mice is compositionally distinct from Jax B6 mice, which likely resulted in different pathological, immunological, and microbial responses to Hp infection.

Purpose-bred common marmosets from domestic sources housed in a US research facility, and used in multiple drug discovery programmes, were noted to have a high incidence of spontaneous inflammatory bowel disease and sporadic cholecystitis and cholangiohepatitis. Inflammatory infiltrates increased in incidence and severity with age. Because Helicobacter spp. have been linked to gastrointestinal diseases, samples from the gastrointestinal tracts of 39 marmosets were screened for Helicobacter spp. by culture and PCR. Helicobacter spp. were frequently detected in marmosets; 28.2% of the marmosets were positive for a proposed novel species, Helicobacter jaachi sp. nov., by culture, and 48.7% were positive by Helicobacter genus-specific PCR. Seventeen strains of Helicobacter sp. from 11 marmosets were cultured from various gastrointestinal sites. Older animals (age 6-11 years) had a higher helicobacter prevalence rate (57.1%) compared with younger animals (age 3-5 years), which had a 27.2% prevalence rate. Cells of H. jaachi sp. nov. were catalase, urease and oxidase positive and had fusiform morphology, with periplasmic fibres and multiple bipolar, sheathed flagella. All isolates had similar 16S and 23S rRNA sequences, which clustered as representatives of a novel Helicobacter species closely related to 'Helicobacter sanguini' (97%), a species isolated from cotton-top tamarins and 'Helicobacter callitrichis' (96%) isolated previously from the faeces of common marmosets. The whole genome sequence of one of the liver isolates, H. jaachi sp. nov. MIT 09-6949(T), had a 1.9 Mb genome length with a 41 mol% DNA G+C content. The type strain of Helicobacter jaachi sp. nov., MIT 09-6949(T), has been deposited in the BCCM/LMG Bacteria Collection as LMG 28613(T). These findings add to the increasing number of animal species with gastrointestinal disease in which novel enterohepatic Helicobacter spp. have been isolated.

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process., National Institutes of Health (U.S.) (grant T32-OD010978-26), National Institutes of Health (U.S.) (grant R01-OD011141), National Institutes of Health (U.S.) (grant R01-DK099204), National Institutes of Health (U.S.) (grant P01-CA028842-29), National Institutes of Health (U.S.) (grant P30-ES002109)

Most genetic variants associated with complex diseases map to non-coding regions of the genome, but determining their phenotypic consequences remains challenging. We devised a bacterial artificial chromosome (BAC) transgenic approach to model the effects of human non-coding single nucleotide polymorphisms (ncSNPs) on complex disease outcomes. As proof-of-concept, we focus on human IL10, a potent anti-inflammatory cytokine and key mediator of gut homeostasis. ncSNPs in the IL10 locus have been strongly associated with a variety of disease risks, including colitis susceptibility, but their biological functions are unclear. We created two genetically humanized IL10BAC transgenic mouse lines based on two common and well-defined −1082/−819/−592 IL10 promoter haplotypes (“ATA” and “GCC”) on a mouse Il10-deficient background. Although both IL10BAC strains were protected from spontaneous colitis, only ATA-bearing mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis, while GCC and WT mice had high rates of morbidity and mortality. We found that ATAs had different kinetics of cell-type-specific IL-10 production pre- and post-DSS treatment compared to GCCs and WTs, which influenced the expression profiles of other cytokines and led to distinct immunophenotypes. The skewed responses culminated in the inability of GCCs and WTs to control DSS-induced bacteremia, resulting in death. Our data suggest that human IL10 ncSNPs impart allele-specific differences in spatiotemporal regulation of IL-10 expression, leading to differential outcomes to experimental colitis. Humanized BAC mice may serve as an important tool in modeling the potential impact of human non-coding variants on complex biological outcomes.

The cytokines IL-10 and IL-22 play important, but presumably distinct roles in promoting intestinal homeostasis. Although the anti-inflammatory actions of IL-10 in the gut are well established, the role(s) of IL-22 remain unclear. Il10−/− mice develop chronic colitis characterized by rectal prolapse, colonic thickening and inflammatory infiltrates, including abundant Th17 cells. We have also observed a modest, but consistent elevation in IL-22 (mRNA and IL-22+CD4 T cells) in colons of Il10−/− mice. This is accompanied by an increased expression of IL-22-target bactericidal genes such as Reg3g, s100a8, and s100a9 and reduced intestinal microbial diversity in Il10−/− mice. To define a potential role for IL-22 in spontaneous colitis, we generated Il10−/− Il22−/− mice by backcrossing. Unexpectedly, concurrent IL-22 deficiency prevented colitis in Il10−/− mice, normalizing histopathology inflammatory scores and restoring microbial diversity to WT levels in Il10−/− Il22−/− mice, despite endemic Helicobacter spp. infection, an established risk factor for colitis in Il10−/− mice. Interestingly, prevention of disease was not accompanied by a reduction in Th17 cells. Importantly, introduction of recombinant IL-22 into the GI tracts of both Il10−/− Il22−/− and WT mice resulted in colonic shortening and thickening after only 2 weeks of treatment, suggesting that IL-22 is at least partially responsible for the immunopathology observed in Il10−/− mice. Thus, although beneficial during certain bacterial infections and in response to acute tissue damage, our data suggest that left unchecked, IL-22 expression can contribute to chronic colitis and appears to be negatively regulated by IL-10.

The draft genome sequences of eight enterohepatic Helicobacter species, H. muridarum, H. trogontum, H. typhlonius, and five unnamed helicobacters, are presented here. Using laboratory mice pervasively infected with helicobacters, we characterized the presence of known virulence factors., National Institutes of Health (U.S.) (grant R01CA067529), National Institutes of Health (U.S.) (grant R01OD011141), National Institutes of Health (U.S.) (grant P01CA26731), National Institutes of Health (U.S.) (grant P30ES002109)

Draft genome sequences of seven enterohepatic Helicobacter species, H. bilis, H. canadensis, H. canis, H. cinaedi, H. winghamensis, H. pullorum, and H. macacae, are presented. These isolates were obtained from clinical patients and a nonhuman primate. Due to potential zoonotic risks, we characterized antibiotic resistance markers and Helicobacter virulence factors., National Institutes of Health (U.S.) (Department of Health and Human Services, under grant U54HG003067), National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.) (Department of Health and Human Services, under grant R01CA067529), National Institutes of Health (U.S.) (Department of Health and Human Services, under grant R01OD011141), National Institutes of Health (U.S.) (Department of Health and Human Services, under grant P01CA26731), National Institutes of Health (U.S.) (Department of Health and Human Services, under grant P30ES002109)

Farnesoid X receptor (FXR) is a nuclear receptor that regulates bile acid metabolism and transport. Mice lacking expression of FXR (FXR KO) have a high incidence of foci of cellular alterations (FCA) and liver tumors. Here, we report that Helicobacter hepaticus infection is necessary for the development of increased hepatitis scores and FCA in previously Helicobacter-free FXR KO mice. FXR KO and wild-type (WT) mice were sham-treated or orally inoculated with H. hepaticus. At 12 months post-infection, mice were euthanized and liver pathology, gene expression, and the cecal microbiome were analyzed. H. hepaticus induced significant increases hepatitis scores and FCA numbers in FXR KO mice (P, National Institutes of Health (U.S.) (NIH R01 OD011141), National Institutes of Health (U.S.) (NIH T32 OD010978), National Institutes of Health (U.S.) (NIH P30 ES002109), National Institutes of Health (U.S.) (P01 CA026731)

The draft genome sequences of six Colombian Helicobacter pylori strains are presented. These strains were isolated from patients from regions of high and low gastric cancer risk in Colombia and were characterized by multilocus sequence typing. The data provide insights into differences between H. pylori strains of different phylogeographic origins., National Institutes of Health (U.S.) (grant P01CA028842), National Institutes of Health (U.S.) (grant P01CA026731), National Institutes of Health (U.S.) (grant P30ES002109)

The discovery of Helicobacter pylori overturned the conventional dogma that the stomach was a sterile organ and that pH values < 4 were capable of sterilizing the stomach. H. pylori are an etiological agent associated with gastritis, hypochlorhydria, duodenal ulcers, and gastric cancer. It is now appreciated that the human stomach supports a bacterial community with possibly 100s of bacterial species that influence stomach homeostasis. Other bacteria colonizing the stomach may also influence H. pylori-associated gastric pathogenesis by creating reactive oxygen and nitrogen species and modulating inflammatory responses. In this review, we summarize the available literature concerning the gastric microbiota in humans, mice, and Mongolian gerbils. We also discuss the gastric perturbations, many involving H. pylori, that facilitate the colonization by bacteria from other compartments of the gastrointestinal tract, and identify risk factors known to affect gastric homeostasis that contribute to changes in the microbiota.

While Helicobacter pylori infects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors in H. pylori pathogenesis, global gene expression of six H. pylori isolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factors cagA , vacA , and babB and were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin of H. pylori strains may promote increased gastric disease.

A novel Helicobacter species Helicobacter japonicum was isolated from the stomach and intestines of clinically normal mice received from three institutes from Japan. The novel Helicobacter sp. was microaerobic, grew at 37°C and 42°C, was catalase and oxidase positive, but urease negative. It is most closely related to the 16S rRNA gene of H.muridarum (98.6%); to the 23S rRNA gene of H.hepaticus (97.9%); to the hsp60 gene of H.typhlonius (87%). The novel Helicobacter sp. has in vitro cytolethal distending toxin (CDT) activity; its cdtB gene sequence has 83.8% identity with that of H.hepaticus The whole genome sequence of H.japonicum MIT 01-6451 has a 2.06-Mb genome length with a 37.5% G + C content. When the organism was inoculated into C57BL/129 IL10-/- mice, it was cultured from the stomach, colon and cecum of infected mice at 6 and 10 weeks post-infection. The cecum had the highest H.japonicum colonization levels by quantitative PCR. The histopathology of the lower bowel was characterized by moderate to severe inflammation, mild edema, epithelial defects, mild to severe hyperplasia, dysplasia and carcinoma. Inflammatory cytokines IFNγ, TNFα and IL17a, as well as iNOS were significantly upregulated in the cecal tissue of infected mice. These results demonstrate that the novel H.japonicum can induce inflammatory bowel disease and carcinoma in IL10-/- mice and highlights the importance of identifying novel Helicobacter spp. especially when they are introduced from outside mouse colonies from different geographic locations.

Helicobacter pylori infection promotes male predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies showed that prophylactic 17β-estradiol (E2) in INS-GAS mice decreases H. pylori–induced carcinogenesis. We examined the effect of E2 and tamoxifen (TAM) on H. pylori–induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks postinfection (WPI), mice were implanted with E2, TAM, both E2 and TAM, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression, and immune cell infiltration were evaluated and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori–infected males receiving E2 and/or TAM, whereas 40% of infected untreated males developed gastric cancer. E2, TAM, and their combination significantly reduced gastric precancerous lesions in infected males compared with infected untreated males (P < 0.001, 0.01, and 0.01, respectively). However, TAM did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or TAM (n = 363 and n = 144, Q < 0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or TAM deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared with controls, E2 decreased gastric mRNA (Q < 0.05) and serum levels (P < 0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P < 0.01). Prevention of H. pylori–induced gastric cancer by E2 and TAM may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts, and downregulation of oncogenic pathways. Cancer Prev Res; 4(9); 1426–35., (Grant R01 AI37750), (Grant R01 CA093405), (Grant P30 ES02109), (Grant P01 CA028842), (Grant T32 RR07036)

Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes ( P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFN γ, IL-17 , TNF α, and iNOS at 6 and 12 mo, as well as H. pylori –specific IgG1 levels at 12 MPI ( P < 0.05) and IgG2c levels at 6 and 12 MPI ( P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI ( P < 0.05) and also than those in infected males at 12 MPI ( P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.

During the past decade, several outbreaks of severe systemic disease associated with Feline calicivirus (FCV) have occurred in the USA and the UK. This new disease has caused high mortality in the affected animals and has been termed virulent systemic (VS)-FCV disease. Currently, there are no genetic or in vitro diagnostic methods to distinguish viruses isolated from cases of VS-FCV disease from other isolates. Here, five in vitro properties, as well as the capsid and proteinase–polymerase (pro–pol) sequences, of a set of FCV isolates that included seven isolates from five distinct VS-FCV outbreaks (‘VS isolates’) were investigated. Although all of the FCV isolates investigated had similar kinetics of growth under single-cycle conditions, VS isolates infected tissue-culture cells more efficiently under multiple-cycle growth conditions. Moreover, it was found that cells infected with VS isolates showed cytopathic effects earlier than cells infected with non-VS isolates, although no difference in relative ATP levels were noted at times when morphological changes were first seen. Both VS- and other (non-VS) isolates of FCV demonstrated similar temperature stabilities. Phylogenetic analyses and alignments of the capsid and pro–pol regions of the genome did not reveal any conserved changes that correlated with virulence, and the VS isolates did not segregate into a unique clade. These results suggest that VS isolates have arisen independently several times since first being described and can spread more efficiently in tissue culture than other isolates when infected at low multiplicity.

Aim Demonstrate the ability of a high-throughput next-generation sequencing (NGS) assay to process 96 samples on the Illumina MiSeq and report results within 72 h. Methods DNA extraction was performed with the BioSprint 96 automatic DNA extractor. PCR products and sequencing library were prepared using an Omixon Holotype X2 kit according to the manufacturer’s instructions. Five loci from 96 samples were sequenced each run using a 300-cycle Illumina MiSeq Reagent v2 kit (150 bp paired end reads). Results We evaluated 40 samples used in the ASHI Proficiency Testing (PT) program in 2012–2014 in either duplicate or triplicate over 2 sequencing runs. Excluding 2 replicate samples due to weak PCR amplification, NGS results were 100% concordant with both our Sanger-based sequencing results and the official ASHI PT results for the 5 loci tested (HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1). For class I genes we provided full coverage from 5’UTR to 3’UTR. For class II genes, coverage was provided from intron 1 to the 3’UTR (DQB1) or intron 4 (DRB1). Specifically looking at the coverage for exons 2 and 3, we had coverage >75× to confidently type the alleles. Furthermore, for samples where results sent to ASHI by different labs were not in full agreement, our NGS results concurred with the interpretation of results provided by ASHI. For illustrative purposes, we present samples HT-148, HT-153, HT-154, HT-168 and HT-175 demonstrating how this NGS methodology provides better calls due to expanded exon coverage and phasing over longer regions. Conclusions Our modified protocol of the Omixon Holotype X2 kit can provide whole gene sequencing data on 5 loci for 96 samples within 72 h of sample delivery. In this study, we evaluated 5 loci and correctly typed 40 ASHI samples in replicate without the need for further trans-ambiguity resolution. Currently, our lab has successfully performed the 7 locus assay including HLA-DPB1 and DQA1 with samples extracted from either blood or buccal swabs with similar high-quality results within 72 h.