The growing popularity of unpasteurized milk in the United States raises public health concerns. We estimated outbreak-related illnesses and hospitalizations caused by the consumption of cow’s milk and cheese contaminated with Shiga toxin–producing Escherichia coli, Salmonella spp., Listeria monocytogenes, and Campylobacter spp. using a model relying on publicly available outbreak data. In the United States, outbreaks associated with dairy consumption cause, on average, 760 illnesses/year and 22 hospitalizations/year, mostly from Salmonella spp. and Campylobacter spp. Unpasteurized milk, consumed by only 3.2% of the population, and cheese, consumed by only 1.6% of the population, caused 96% of illnesses caused by contaminated dairy products. Unpasteurized dairy products thus cause 840 (95% CrI 611–1,158) times more illnesses and 45 (95% CrI 34–59) times more hospitalizations than pasteurized products. As consumption of unpasteurized dairy products grows, illnesses will increase steadily; a doubling in the consumption of unpasteurized milk or cheese could increase outbreak-related illnesses by 96%.

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The metabolism of tryptophan to nicotinamide adenine dinucleotide (NAD+) through the kynurenine pathway is increasingly linked to aging and age-associated disease. Kynurenine pathway enzymes and metabolites influence a range of molecular processes critical to healthy aging, including regulation of inflammatory and immune responses, cellular redox homeostasis, and energy production. Aberrant kynurenine metabolism is observed during normal aging and has been implicated in a range of age-associated pathologies, including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. In previous work, we and others identified three genes-kynu-1, tdo-2, and acsd-1-encoding kynurenine pathway enzymes for which decreasing expression extends lifespan in invertebrate models. Here we report that knockdown of haao-1, a fourth kynurenine pathway gene encoding the enzyme 3-hydroxyanthranilic acid dioxygenase (HAAO), extends lifespan by ~30% and delays age-associated decline in health in Caenorhabditis elegans. This lifespan extension is mediated by increased physiological levels of the HAAO substrate 3-hydroxyanthranilic acid (3HAA). 3HAA increases resistance to oxidative stress during aging by directly degrading hydrogen peroxide and activating the Nrf2/SKN-1 oxidative stress response. Aging mice fed a diet supplemented with 3HAA are similarly long-lived. Our results identify HAAO and 3HAA as novel therapeutic targets for age-associated disease. This works provides a foundation for more detailed examination of the molecular mechanisms underlying the benefits of 3HAA, and how these mechanisms interact with other interventions both within and beyond the kynurenine pathway. We anticipate that these findings will bolster growing interest in developing pharmacological strategies to target tryptophan metabolism to improve health aging.

Caenorhabditis elegans are an important model system for host-microbe research due to the ability to rapidly quantify the influence of microbial exposure on whole-organism survival and rapidly quantify microbial load. To date, the majority of host-pathogen interaction studies rely on host group survival and cross-sectional examination of infection severity. Here we present a new system called Systematic Imaging of Caenorhabditis Killing Organisms (SICKO) capable of characterizing longitudinal interactions between host and pathogens in individual C. elegans, enabling researchers to capture dynamic changes in gut colonization between individuals and quantify the impact of bacterial colonization events on host survival. Using this system, we demonstrate that gut colonization by the strain of Escherichia coli used as a common laboratory food source dramatically impacts the lifespan of C. elegans. Additionally, we show that immunodeficient animals, lacking the pmk-1 gene, do not significantly alter the progression of bacterial infection, but rather suffer an increased rate of gut colony initiation. This new system provides a powerful tool into understanding underlying mechanisms of host-microbe interaction, opening a wide avenue for detailed research into therapies that combat pathogen induced illness, the benefits imparted by probiotic bacteria, and understanding the role of the microbiome in host health .

Introduction Mutant GOF p53 is an epigenetic regulator which promotes oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim−) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim−) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knockdown GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression. Methods Proliferation rate, MMP expression, and migration of SW13(Vim−), SW13(Vim+), shRNA SW13(Vim−), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knockdown by shRNA. Proteins were identified by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively. Results Data suggest that there is a shift from planar rafts in the SW13(Vim−) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knockdown increases the proliferation rate in SW13(Vim−) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knockdown decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim−). Conclusions Our data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro oncogenic properties of GOF p53, may do so, in part, by altering raft composition and signaling.

Aging is characterized by a progressive decline in the normal physiological functions of an organism, ultimately leading to mortality. Metabolic changes throughout the aging process disrupt the balance and homeostasis of the cell. The kynurenine metabolic pathway is the sole de novo biosynthetic pathway for producing NAD+ from ingested tryptophan. Altered kynurenine pathway activity is associated with both aging and a variety of age-associated diseases, and kynurenine-based interventions can extend lifespan in Caenorhabditis elegans. Our laboratory recently demonstrated knockdown of the kynurenine pathway enzymes kynureninase (KYNU) or 3-hydroxyanthranilic acid dioxygenase (HAAO) increases lifespan by 20-30% in C elegans. However, the mechanism of how these interventions may modulate response against different stressors during the aging process has yet to be explored. Fluorescent reporter strains show the stress-responsive transcription factors skn-1 (ortholog of NRF2/NFE2L2; oxidative stress response) and hif-1 (ortholog of HIF1A; hypoxic stress response) to be highly upregulated when the kynurenine pathway is inhibited. We also demonstrated the increase expression of gst-4 and gcs-1 (transcriptional targets skn-1), which are involved in production of the antioxidant glutathione (GSH), as well as upregulation of cysl-2 (transcriptional target of hif-1), a regulator of cysteine biosynthesis from serine. We hypothesize that lifespan extension resulting from kynurenine pathway inhibition is mediated, at least in part, by upregulation of these transcription factors, providing elevated defense against oxidative stress and hypoxic stress.

Aberrant kynurenine pathway metabolism is increasingly linked to aging and age-associated disease. Kynurenine metabolic activity increases with age and becomes dysregulated during various forms of age-associated pathology in humans. By manipulating one or more kynurenine pathway enzymes and metabolites, we have extended lifespan up to 40% in Caenorhabditis elegans. In particular, elevating physiological levels of the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3HAA) by directly supplementing 3HAA or inhibiting the enzyme 3HAA dioxygenase (HAAO) extends C. elegans lifespan by ~30%. 3HAA delivered chronically in chow similarly extends lifespan in aged C57BL/6 mice. In ongoing work, we are investigating the mechanisms underlying the benefits of multiple kynurenine pathway interventions using tools in C. elegans, mice, and human cell culture. We have preliminary evidence for activation of broad-spectrum cellular stress response, enhanced immune function, and reduced inflammation. Among other roles, the kynurenine pathway is the sole metabolic route for de novo synthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan in Eukaryotic cells. We are examining the regulatory interaction between kynurenine metabolism and the two NAD+ recycling pathways, Salvage and Preiss-Handler, both as potential mechanistic mediators and as possible parallel targets for combined interventions with synergistic benefits in aging. We are further evaluating the impact of these interventions in several models of specific age-associated diseases, including sepsis, chronic inflammation, stroke, Alzheimer’s disease, and cancer. Finally, we are developing pharmaceutical strategies to replicate key genetic and metabolic interventions within the kynurenine pathway that can be readily translated into clinical applications.

The purpose of the PNRP patient navigation is to provide individualized assistance to persons with abnormal cancer screening tests or cancer diagnosis by trained, culturally sensitive navigators familiar with the patient's community. The Moffitt Cancer Center is one of nine NCI-funded sites nationally to study the impact that patient navigation has on four main outcomes: 1) time from screening to definitive diagnosis; 2) time from definitive diagnosis to treatment; 3) patient satisfaction; and 4) cost effectiveness. Moffitt's patient navigators navigated over 640 patients referred from seven community clinics in four counties. The majority of the patients were female (93%), Hispanics (54%), with annual household income under $20,000 (80%), mostly unemployed (47.5%), and with an average of 8.8 years of education. Lay patient navigators familiar with the patients’ culture and language were trained to navigate patients with breast or colorectal cancer screening abnormalities. The navigators were asked to record in a log the barriers that they identified, along with the actions they took to assist the patients address such barriers. Navigators addressed 20 different types of barriers in an average of 10 encounters per patient. This presentation will 1) describe the processes of patient navigation utilized by Moffitt PNRP, including training requirements, and caseloads for patient navigators, and 2) discuss the most relevant barriers that navigators found among medically underserved patients to access cancer health services, and actions taken to address such barriers. Moffitt patient navigators engage in a number of actions to help address unique barriers faced by this population when trying to access cancer care services. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A31.