Adam S. Kim

Clostridium difficile toxins A and B can alter epithelial permeability and promote bacterial paracellular migration through HT-29 enterocytes.

Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis. We hypothesized that C. difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier. Experiments were designed to clarify the effects of C. difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions. In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL). To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles. Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers. To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers. Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes. Thus intestinal colonization with toxigenic C. difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.

Hypoxia and extraintestinal dissemination of Candida albicans yeast forms.

Candida albicans is a pleomorphic fungus with budding yeast and filamentous forms, and is a frequent cause of complicating infections in patients who are postsurgical, in shock, and have trauma. Many cases of systemic candidiasis are thought to originate from the intestine, but it is unclear if the filament or the yeast is the more invasive form. Because C. albicans is relatively noninvasive and because mesenteric ischemia is thought to facilitate extraintestinal microbial dissemination, wild-type C. albicans CAF2 and mutant HLC54 (defective in filament formation) were orally inoculated into antibiotic-treated mice that were housed exclusively in room air, or were intermittently exposed to 10% oxygen for 1-h intervals. Both strains of C. albicans colonized the cecum in similar numbers (approximately 106.7/g). C. albicans translocation to the draining mesenteric lymph nodes was not detected in mice inoculated with CAF2 (normoxic or hypoxic) or in normoxic mice inoculated with HLC54, but was detected in 33% (P < 0.01) of hypoxic mice inoculated with HLC54. Using Caco-2 and HT-29 enterocytes cultivated on plastic dishes and pretreated for 48 h in 10% oxygen, adherence of C. albicans HLC54 was decreased compared with wild-type CAF2, and hypoxia had no noticeable effect on adherence of either CAF2 or HLC54. Using enterocytes cultivated on permeable 8-&mgr;m filters, transepithelial migration of C. albicans CAF2 and HLC54 appeared similar. Thus, C. albicans HLC54 (defective in filament formation) was more invasive in hypoxic mice compared with wild-type CAF2, and host factors (e.g., mesenteric ischemia) rather than an innate ability to interact with enterocytes might play a more important role in extraintestinal dissemination of C. albicans yeast forms.

Attenuated Salmonella typhimurium prevents the establishment of unresectable hepatic metastases and improves survival in a murine model

PURPOSE The authors investigated the utility of attenuated Salmonella typhimurium for preventing the establishment of hepatic metastases in a murine model. METHODS A single, oral 10(8) cfu dose of attenuated S typhimurium was given 8 days before the establishment of a model of unresectable hepatic metastases. Animals were assessed for hepatic tumor number and volume, hepatic lymphocyte population analysis, and survival. RESULTS Pretreatment with Salmonella provided a 10-fold reduction in hepatic tumor burden compared with saline-treated controls. The antitumor effect is associated with markedly elevated natural killer (NK), CD8+ and CD4+ hepatic lymphocytes. Pretreatment with Salmonella provided a 90-day survival rate of 30%, whereas control animals were dead by 30 days. All long-term survivors were devoid of hepatic tumor. CONCLUSIONS Attenuated S typhimurium effectively prevents the establishment of hepatic metastases in a murine model, providing a clear survival benefit. Thus, it may represent a novel form of in vivo immunotherapy for the prevention of hepatic metastases for patients with locally advanced colorectal cancer.

Cyclooxygenase-2 Inhibition Augments the Hepatic Antitumor Effect of Oral Salmonella Typhimurium in a Model of Mouse Metastatic Colon Cancer

AbstractINTRODUCTION: Oral inoculation with a nontoxic, attenuated strain of Salmonella typhimurium reduces tumor burden and improves survival in a mouse model of metastatic colon cancer. These effects are likely mediated by S. typhimurium-induced increases in hepatic natural killer leukocytes. Cyclooxygenase-2 inhibitors may mediate antitumor effects through antiangiogenic, immune, or proapoptotic pathways. We hypothesized that cyclooxygenase-2 inhibitors would act synergistically with S. typhimurium, resulting in additional antitumor effects. METHODS: Four groups of mice were studied: control, S. typhimurium alone, cyclooxygenase-2 inhibitor alone, and S. typhimurium plus cyclooxygenase-2 inhibitor. Mice were given normal drinking water (control, S. typhimurium alone) or water with 1,600 parts per million cyclooxygenase-2 inhibitor (cyclooxygenase-2 inhibitor alone, and S. typhimurium plus cyclooxygenase-2 inhibitor) and orally inoculated with saline (control, cyclooxygenase-2 inhibitor alone) or 109 S. typhimurium (S. typhimurium alone, S. typhimurium plus cyclooxygenase-2 inhibitor). Twenty-four hours later, all mice underwent laparotomy, and 5 × 104 MCA38 murine adenocarcinoma cells were injected into the spleen. On Day 14, hepatic tumor number and tumor volume was quantitated and hepatic leukocytes were analyzed by flow cytometry. RESULTS: Compared with control mice orally inoculated with saline, S. typhimurium-treated mice had fewer and smaller tumors; mice treated with cyclooxygenase-2 inhibitor alone had tumor burden similar to control mice, and mice treated with S. typhimurium plus cyclooxygenase-2 inhibitor had fewer and smaller tumors compared with all other groups. Increased liver natural killer cells and decreased CD4+ and CD8+ T cells were observed in both S. typhimurium-treated groups. No alterations in hepatic leukocyte phenotype were observed in mice receiving cyclooxygenase-2 inhibitor alone. CONCLUSION: Oral cyclooxygenase-2 inhibitor appeared to act synergistically with S. typhimurium to reduce tumor burden. This combination therapy may have clinical application in the treatment or prevention of hepatic metastases associated with colorectal cancer.

207 Developing Genetic Models for Predicting Crohn's Disease Risk

primary clusters. One cluster included all but one non-IBD sample as well as ~2/3 of UC and ~3/4 of CD samples, and was classified for the purposes of this study as normal. The second cluster, which included the remaining CD and UC samples, exhibited depletion of Firmicutes and Bacteroidetes sequences, and was classified for the purposes of this study as dysbiosis. The odds ratio of detecting dysbiosis in a NOD2R IBD patient was 3.7 (1.3-11, P = 0.018). Application of the Unifrac test indicated that for CD patients, NOD2R is significantly associated with altered intestinal microbiota, relative to NOD2S. CONCLUSIONS: These results support the hypothesis that NOD2 risk alleles contribute to the microbial community imbalances observed in a subset of IBD patients.