Nicola Parry

Stressor Exposure Disrupts Commensal Microbial Populations in the Intestines and Leads to Increased Colonization by Citrobacter rodentium

ABSTRACT The gastrointestinal tract is colonized by an enormous array of microbes that are known to have many beneficial effects on the host. Previous studies have indicated that stressor exposure can disrupt the stability of the intestinal microbiota, but the extent of these changes, as well as the effects on enteric infection, has not been well characterized. In order to examine the ability of stressors to induce changes in the gut microbiota, we exposed mice to a prolonged restraint stressor and then characterized microbial populations in the intestines using both traditional culture techniques and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Exposure to the stressor led to an overgrowth of facultatively anaerobic microbiota while at the same time significantly reducing microbial richness and diversity in the ceca of stressed mice. Some of these effects could be explained by a stressor-induced reduction in the relative abundance of bacteria in the family Porphyromonadaceae. To determine whether these alterations would lead to increased pathogen colonization, stressed mice, as well as nonstressed controls, were challenged orally with the enteric murine pathogen Citrobacter rodentium. Exposure to the restraint stressor led to a significant increase in C. rodentium colonization over that in nonstressed control mice. The increased colonization was associated with increased tumor necrosis factor alpha (TNF-α) gene expression in colonic tissue. Together, these data demonstrate that a prolonged stressor can significantly change the composition of the intestinal microbiota and suggest that this disruption of the microbiota increases susceptibility to an enteric pathogen.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer

Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 μM. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications.

Piglet Models of Acute or Chronic Clostridium difficile Illness

We examined the piglet model of Clostridium difficile illness (CDI) in humans, because swine are naturally susceptible to C. difficile. The piglet is a reproducible model of acute or chronic CDI with characteristic pseudomembranous colitis. Germ-free piglets were consistently and extensively colonized after oral challenge with the human strain 027/BI/NAP1, establishing an infectious dose-age relationship. This allowed a demarcation between acute fatal and chronic models. The clinical manifestations of disease inclusive of gastrointestinal and systemic symptoms and characteristic mucosal lesions of the large bowel (including pseudomembranous colitis) are described. Additionally, we demonstrate the presence of toxins in feces, body fluids, and serum and a significant elevation in interleukin 8 levels in animals with severe disease. We conclude that piglets infected with C. difficile mimic many of the key characteristics observed in humans with CDI and are suitable animals in which to investigate the role played by virulence attributes, drug efficacy, and vaccine candidates.

Antibody Against TcdB, but Not TcdA, Prevents Development of Gastrointestinal and Systemic Clostridium difficile Disease

BACKGROUND A dramatic increase in morbidity and mortality from Clostridium difficile infection (CDI) due to the recent emergence of virulent, antibiotic-resistant strains has led to a search for alternatives to antibiotics, including vaccines and immune-based therapy that target the 2 key toxins-TcdA and TcdB. METHODS We investigated the efficacy of specific human monoclonal antibodies (HuMab) and alpaca polyclonal antibodies against each toxin separately and in combination in the gnotobiotic piglet model of CDI. Additionally, the HuMab and polyclonal antibodies were exploited to investigate the precise contribution of each toxin to systemic and/or gastrointestinal (GI) tract disease. RESULTS Our results indicate that TcdB is an important virulence factor associated with GI and systemic pathology. Administration of anti-TcdB antibody alone or with anti-TcdA protected 100% of piglets from development of systemic CDI and minimized GI lesions. Conversely, 100% of piglets administered only anti-TcdA developed severe GI and systemic disease, with 67%-83% fatality, faring worse than placebo-treated control animals. CONCLUSIONS These results highlight the importance of TcdB in the pathogenesis of CDI and the effectiveness of TcdB-specific antibody in treating CDI. However, the results raise new questions regarding the nature of TcdA interaction with therapeutic antibodies.

Gastric Helicobacter species as a cause of feline gastric lymphoma: A viable hypothesis

Gastric Helicobacter spp. are associated with chronic inflammation and neoplastic transformation in humans as well as domestic and laboratory species. The present study examined the association of Helicobacter heilmannii (Hhe) infection in pet cats with feline gastric mucosa associated lymphoid tissue (MALT) lymphoma. Tissues were collected via gastric biopsy or at necropsy from 47 pet cats with clinical signs of gastrointestinal disease, including vomiting and inappetance, and classified as gastritis (14/47), lymphoma (31/37), or normal (2/47). Tissues positive for argyrophilic organisms with Warthin-Starry stain (29/47) were assessed by fluorescent in situ hybridization (FISH) for the presence of Hhe strains 1-4 as well as with a fifth probe that detected Helicobacter salomonis, Helicobacter bizzozeronii, or Helicobacter felis. A significant association of positive Warthin-Starry status with Hhe infection was found in cases of sick cats (22/29; p<0.05 by Chi-square; chi(2)=7.034). Interestingly, a significant association between Hhe status and a diagnosis of lymphoblastic or lymphocytic lymphoma was observed as well in a subset of 24 Warthin-Starry positive lymphoma cases: of lymphoblastic lymphoma cases, 13/17 were positive for Hhe (p<0.05; chi(2)=4.854). Hhe strains 2 and 4 were most commonly found (18/29 and 17/29, respectively) among sick cats, although a higher than expected number of cats was also positive for Hhe1, which initial reports have described as rare in cats and common in humans. The association found between a positive Hhe status with the presence of feline gastric lymphoma, especially lymphoblastic lymphoma, argues for the need to conduct prospective studies to better identify the frequency and strain distribution of Hhe infection in both healthy and clinically ill cats, particularly those cats with gastric lymphoma.

Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection

ABSTRACT Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus, which may contribute to stressor-enhanced C. rodentium-induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium-induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium-enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium. Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection.

17β-Estradiol and Tamoxifen Prevent Gastric Cancer by Modulating Leukocyte Recruitment and Oncogenic Pathways in Helicobacter Pylori–Infected INS-GAS Male Mice

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.

Gnotobiotic piglet infection model for evaluating the safe use of antibiotics against Escherichia coli O157:H7 infection

BACKGROUND Shiga toxin (Stx)-producing Escherichia coli (STEC), especially O157:H7, cause bloody diarrhea, and in 3%-15% of individuals the infection leads to hemolytic uremic syndrome (HUS) or other complications. Use of antibiotics to treat STEC infections is controversial. Here, we describe the use of piglets to evaluate the efficacy and mechanism of action of antibiotics in these infections. METHODS The effects of 2 antibiotics on STEC toxin production and their mechanisms of action were first determined by enzyme-linked immunosorbent assay and subsequently evaluated clinically in the gnotobiotic piglet infection model. RESULTS In vitro treatment of clinical and isogenic strains with ciprofloxacin increased the production of Stx2 via phage induction but not the production of Stx1. Azithromycin caused no significant increase in toxin production. After treatment with ciprofloxacin, infected piglets had diarrhea and the severe fatal neurological symptoms associated with Stx2 intoxication. Characteristic petechial hemorrhages in the cerebellum were more severe in ciprofloxacin-treated animals than in control animals. In contrast, azithromycin-treated piglets survived the infection and had little or no brain hemorrhaging. CONCLUSIONS The increased in vitro toxin production caused by ciprofloxacin was strongly correlated with death and an increased rate of cerebellar hemorrhage, in contrast to the effect of azithromycin. The piglet is a suitable model for determining the effectiveness and safety of antibiotics available to treat patients.

Gut microbiome phenotypes driven by host genetics affect arsenic metabolism.

Large individual differences in susceptibility to arsenic-induced diseases are well-documented and frequently associated with different patterns of arsenic metabolism. In this context, the role of the gut microbiome in directly metabolizing arsenic and triggering systemic responses in diverse organs raises the possibility that gut microbiome phenotypes affect the spectrum of metabolized arsenic species. However, it remains unclear how host genetics and the gut microbiome interact to affect the biotransformation of arsenic. Using an integrated approach combining 16S rRNA gene sequencing and HPLC-ICP-MS arsenic speciation, we demonstrate that IL-10 gene knockout leads to a significant taxonomic change of the gut microbiome, which in turn substantially affects arsenic metabolism.