Novel Mouse Models of Bladder Cancer Identify a Prognostic Signature Associated with Risk of Disease Progression.

To study the progression of bladder cancer from non-muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. SIGNIFICANCE: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations.

Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.

Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways.

The importance of estrogens in the etiology of breast cancer is widely recognized. Estrogen-induced oxidative stress has been implicated in this carcinogenic process. Resveratrol (Res), a natural antioxidant phytoestrogen has chemopreventive effects against a variety of illnesses including cancer. The objective of the present study was to characterize the mechanism(s) of Res-mediated protection against estrogen-induced breast carcinogenesis. Female August Copenhagen Irish rats were treated with 17ß-estradiol (E2), Res and Res + E2 for 8 months. Cotreatment of rats with Res and E2 inhibited E2-mediated proliferative changes in mammary tissues and significantly increased tumor latency and reduced E2-induced breast tumor development. Resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues. Expression of NRF2-regulated antioxidant genes NQO1, SOD3 and OGG1 that are involved in protection against oxidative DNA damage was increased in Res- and Res + E2-treated mammary tissues. Resveratrol also prevented E2-mediated inhibition of detoxification genes AOX1 and FMO1. Inhibition of E2-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after Res treatment indicated Res-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis. Resveratrol treatment also induced apoptosis and inhibited E2-mediated increase in DNA damage in mammary tissues. Increased apoptosis and decreased DNA damage, cell migration, colony and mammosphere formation in Res- and Res + E2-treated MCF-10A cells suggested a protective role of Res against E2-induced mammary carcinogenesis. Small-interfering RNA-mediated silencing of NRF2 inhibited Res-mediated preventive effects on the colony and mammosphere formation. Taken together, these results suggest that Res inhibits E2-induced breast carcinogenesis via induction of NRF2-mediated protective pathways.

Pharmacokinetics of fluoxetine in pregnant baboons (Papio spp.).

Fluoxetine is used to treat a number of psychiatric conditions in humans and behavioral problems in animals. Its use in pregnancy must balance maternal benefit with potential risk to the fetus. Knowledge of adult and fetal drug disposition can assist clinicians in selecting therapy that minimizes adverse effects to the fetus. Nonhuman primate models are used frequently in drug dose-translation studies, and pregnancy in baboons has many similarities to human pregnancy. Accordingly, pharmacokinetic analysis of a series of fluoxetine and norfluoxetine administrations to pregnant baboons was performed. The mean maternal baboon steady-state clearance of fluoxetine (42 mL/min/kg) was considerably higher than that in humans. Norfluoxetine, the major active metabolite, had a higher metabolite-to-drug ratio (8.7) than that found in humans, particularly with oral dosing. These results are consistent with more extensive metabolism in baboons than in humans and leads to a higher clearance than would be expected from allometric scaling. Fetal-to-maternal fluoxetine and norfluoxetine ratios under steady-state conditions were similar to those in humans, with fetal concentrations of fluoxetine 42% and norfluoxetine 47% of maternal concentrations. The fetal clearance of fluoxetine (303 ± 176 mL/min) and norfluoxetine (450 mL/min) exceeded reported placental blood flow. Understanding these species-associated differences in metabolism is a prerequisite to extrapolating data between species. Nonetheless, nonhuman primates are likely to remain valuable models for pharmacokinetic studies during pregnancy, particularly those directed toward fetal neurodevelopmental effects. Our results also are applicable to determining appropriate dosing of nonhuman primates in clinical settings.

Partial small bowel resection with sleeve gastrectomy increases adiponectin levels and improves glucose homeostasis in obese rodents with type 2 diabetes.

BACKGROUND: The aim of this study was to examine the effect of small bowel resection with and without sleeve gastrectomy on glucose homeostasis in an obese rodent model of type 2 diabetes. METHODS: Zucker diabetic fatty rats were randomized into three surgical groups: Sham, small bowel resection, and small bowel resection with sleeve gastrectomy (BRSG). Weight and fasting glucose levels were measured at randomization and monitored after surgery. Oral glucose tolerance testing was performed at baseline and 45 days after surgery to assess glucose homeostasis and peptide changes. RESULTS: At baseline, all animals exhibited impaired glucose tolerance and showed no difference in weight or fasting (area under the curve) AUC(glucose). At sacrifice, Sham animals weighed more than BRSG animals (p = 0.047). At day 45, the Sham group experienced a significant increase in AUC(glucose) compared to baseline (p = 0.02), whereas there was no difference in AUC(glucose) in either surgical group at any time point: BR (p = 0.58) and BRSG (p = 0.56). Single-factor ANOVA showed a significant difference in AUC(glucose) of p = 0.004 between groups postoperatively: Sham (50,745 ± 11,170) versus BR (23,865 ± 432.6) (p = 0.01); Sham versus BRSG (28,710 ± 3188.8) (p = 0.02). There was no difference in plasma insulin, GLP-1, or adiponectin levels before surgery, although 45 days following surgery adiponectin levels where higher in the BRSG group (p = 0.004). CONCLUSIONS: Partial small bowel resection improved glucose tolerance independent of weight. The combination of small bowel resection and sleeve gastrectomy leads to an increase in adiponectin levels, which may contribute to improved glucose homeostasis.

Bloodstream infections in patients given treatment with intravenous prostanoids.

OBJECTIVE: In September 2006, the Centers for Disease Control and Prevention was notified of cases of gram-negative bloodstream infection (BSI) occurring among outpatients who received an intravenous formulation of the prostanoid treprostinil. An investigation was conducted to determine rates of prostanoid-associated BSI in this patient population and possible risk factors for infection. METHODS: We performed a retrospective cohort study of patients who had received intravenous formulations of at least 1 of the 2 approved prostanoids (epoprostenol and treprostinil) from January 1, 2004, through late 2006. Chart reviews were conducted at 2 large centers for pulmonary arterial hypertension, and a survey of infection control practices was conducted at 1 center. RESULTS: A total of 224 patients were given intravenous prostanoid treatment, corresponding to 146,093 treatment-days during the study period. Overall, there were 0.55 cases of BSI and 0.18 cases of BSI due to gram-negative organisms per 1,000 treatment-days. BSI rates were higher for patients who received intravenous treprostinil than for patients who received intravenous epoprostenol (1.13 vs. 0.42 BSIs per 1,000 treatment-days; P < .001), as were rates of BSI due to gram-negative organisms (0.81 vs. 0.04 BSIs per 1,000 treatment-days; P < .001). Adjusted hazard ratios for all BSIs and for BSIs due to gram-negative organisms were higher among patients given treatment with intravenous treprostinil. The survey identified no significant differences in medication-related infection control practices. CONCLUSION: At 2 centers, BSI due to gram-negative pathogens was more common than previously reported and was more frequent among patients given treatment with intravenous treprostinil than among patients given treatment with intravenous epoprostenol. Whether similar results would be found at other centers for pulmonary arterial hypertension warrants further investigation. This investigation underscores the importance of surveillance and evaluation of healthcare-related adverse events in patients given treatment primarily as outpatients.

Outbreak of Enterococcus gallinarum infections after total knee arthroplasty.

In September 2006, we investigated a cluster of 9 patients who developed Enterococcus gallinarum infection after total knee arthroplasty. Isolates recovered from these patients were from the same outbreak strain. Although all 9 patients were monitored by the same healthcare personnel, were given spinal anesthesia, and had the same specific type of wound irrigation procedure performed during their hospitalization, the source or sources of these infections were not identified.