Hypoxia-Inducible Factor α Subunits Regulate Tie2-Expressing Macrophages That Influence Tumor Oxygen and Perfusion in Murine Breast Cancer.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

The Next Generation of Dietitians: Implementing Dietetics Education and Practice in Integrative Medicine.

Integrative medicine is a quickly expanding field of health care that emphasizes nutrition as a key component. Dietitians and nutritionists have an opportunity to meet workforce demands by practicing dietetics and integrative medicine (DIM). The purpose of this article is to describe a DIM education program and practicum. We report the results of an interprofessional nutrition education and practicum program between the University of Kansas Medical Center (KUMC) Department of Dietetics and Nutrition and KU Integrative Medicine. This partnered program provides training that builds on the strong foundation of the Nutrition Care Process and adds graduate-level educational and practicum experiences in foundational integrative medicine knowledge, including nutritional approaches from a systems biology perspective, nutrigenomics, and biochemistry as the core knowledge to understand the root cause of a chronic disorder and to choose appropriate nutritional tools for interventions. This interprofessional KUMC program provides a dietetic internship, master's degree, and graduate certificate in DIM and fulfills a need for dietitians and nutritionists who seek careers practicing in an integrative medicine setting. The program fulfills expanding workforce needs to provide quality health care for patients with chronic illnesses.

Nutritional and Metabolic Biomarkers in Autism Spectrum Disorders: An Exploratory Study.

CONTEXT: Autism spectrum disorder (ASD) is currently on the rise, now affecting approximately 1 in 68 children in the United States according to a 2010 surveillance summary from the Centers for Disease Control and Prevention (CDC). This figure is an estimated increase of 78% from the figure in 2002. The CDC suggests that more investigation is needed to understand this astounding increase in autism in such a short period. OBJECTIVE: The aim of this pilot study was to determine whether a group of children with ASD exhibited similar variations in a broad array of potential correlates, including medical histories, symptoms, genetics, and multiple nutritional and metabolic biomarkers. DESIGN: This study was a retrospective, descriptive chart review. SETTING: The study took place at the University of Kansas Medical Center (KUMC). PARTICIPANTS: Participants were 7 children with ASD who had sought treatment at the Integrative Medicine Clinic at the medical center. RESULTS: A majority of the children exhibited an elevated copper:zinc ratio and abnormal vitamin D levels. Children also demonstrated abnormal levels of the essential fatty acids: (1) α-linolenic acid (ALA)- C13:3W3, and (2) linoleic acid (LA)-C18:2W6; high levels of docosahexaenoic acid (DHA); and an elevated ω-6:ω-3 ratio. Three of 7 children demonstrated abnormal manganese levels. Children did not demonstrate elevated urine pyruvate or lactate but did have abnormal detoxification markers. Three of 7 patients demonstrated abnormalities in citric acid metabolites, bacterial metabolism, and fatty acid oxidation markers. A majority demonstrated elevated serum immunoglobulin G (IgG) antibodies to casein, egg whites, egg yolks, and peanuts. A majority had absent glutathione S-transferase (GSTM) at the 1p13.3 location, and 3 of 7 children were heterozygous for the glutathione S-transferase I105V (GSTP1). A majority also exhibited genetic polymorphism of the mitochondrial gene superoxide dismutase A16V (SOD2). CONCLUSIONS: The findings from this small group of children with ASD points to the existence of nutritional, metabolic, and genetic correlates of ASD. These factors appear to be important potential abnormalities that warrant a case control study to evaluate their reliability and validity as markers of ASD.

Involvement of tumor macrophage HIFs in chemotherapy effectiveness: mathematical modeling of oxygen, pH, and glutathione.

The four variables, hypoxia, acidity, high glutathione (GSH) concentration and fast reducing rate (redox) are distinct and varied characteristics of solid tumors compared to normal tissue. These parameters are among the most significant factors underlying the metabolism and physiology of solid tumors, regardless of their type or origin. Low oxygen tension contributes to both inhibition of cancer cell proliferation and therapeutic resistance of tumors; low extracellular pH, the reverse of normal cells, mainly enhances tumor invasion; and dysregulated GSH and redox potential within cancer cells favor their proliferation. In fact, cancer cells under these microenvironmental conditions appreciably alter tumor response to cytotoxic anti-cancer treatments. Recent experiments measured the in vivo longitudinal data of these four parameters with tumor development and the corresponding presence and absence of tumor macrophage HIF-1α or HIF-2α in a mouse model of breast cancer. In the current paper, we present a mathematical model-based system of (ordinary and partial) differential equations to monitor tumor growth and susceptibility to standard chemotherapy with oxygen level, pH, and intracellular GSH concentration. We first show that our model simulations agree with the corresponding experiments, and then we use our model to suggest treatments of tumors by altering these four parameters in tumor microenvironment. For example, the model qualitatively predicts that GSH depletion can raise the level of reactive oxygen species (ROS) above a toxic threshold and result in inhibition of tumor growth.

Macrophage colony-stimulating factor augments Tie2-expressing monocyte differentiation, angiogenic function, and recruitment in a mouse model of breast cancer.

Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.

Pediatric Integrative Medicine Approaches to Attention Deficit Hyperactivity Disorder (ADHD).

Attention deficit hyperactivity disorder (ADHD) is the most common neuropsychiatric disorder in children and is increasing in prevalence. There has also been a related increase in prescribing stimulant medication despite some controversy whether ADHD medication makes a lasting difference in school performance or achievement. Families who are apprehensive about side effects and with concerns for efficacy of medication pursue integrative medicine as an alternative or adjunct to pharmacologic and cognitive behavioral treatment approaches. Integrative medicine incorporates evidence-based medicine, both conventional and complementary and alternative therapies, to deliver personalized care to the patient, emphasizing diet, nutrients, gut health, and environmental influences as a means to decrease symptoms associated with chronic disorders. Pediatric integrative medicine practitioners are increasing in number throughout the United States because of improvement in patient health outcomes. However, limited funding and poor research design interfere with generalizable treatment approaches utilizing integrative medicine. The use of research designs originally intended for drugs and procedures are not suitable for many integrative medicine approaches. This article serves to highlight integrative medicine approaches in use today for children with ADHD, including dietary therapies, nutritional supplements, environmental hygiene, and neurofeedback.

Hypoxia-inducible factor-2α regulates GM-CSF-derived soluble vascular endothelial growth factor receptor 1 production from macrophages and inhibits tumor growth and angiogenesis.

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O(2) secrete high levels of a soluble form of the VEGF receptor 1 (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using small interfering RNA targeting to deplete hypoxia-inducible factor (HIF)-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O(2) was dependent on HIF-2α, whereas HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1α(flox/flox)- or HIF-2α(flox/+)/LysMcre mice). GM-CSF treatment increased intratumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1α(flox/flox)/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared with control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1α(flox/flox)/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2α(flox/+)/LysMcre mice, and, correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.

Low Voltage Electrowetting-on-Dielectric Platform using Multi-Layer Insulators.

A low voltage, two-level-metal, and multi-layer insulator electrowetting-on-dielectric (EWD) platform is presented. Dispensing 300pl droplets from 140nl closed on-chip reservoirs was accomplished with as little as 11.4V solely through EWD forces, and the actuation threshold voltage was 7.2V with a 1Hz voltage switching rate between electrodes. EWD devices were fabricated with a multilayer insulator consisting of 135nm sputtered tantalum pentoxide (Ta(2)O(5)) and 180nm parylene C coated with 70nm of CYTOP. Furthermore, the minimum actuation threshold voltage followed a previously published scaling model for the threshold voltage, V(T), which is proportional to (t/ε(r))(1/2), where t and ε(r) are the insulator thickness and dielectric constant respectively. Device threshold voltages are compared for several insulator thicknesses (200nm, 500nm, and 1µm), different dielectric materials (parylene C and tantalum pentoxide), and homogeneous versus heterogeneous compositions. Additionally, we used a two-level-metal fabrication process, which enables the fabrication of smaller and denser electrodes with high interconnect routing flexibility. We also have achieved low dispensing and actuation voltages for scaled devices with 30pl droplets.

NudC is required for Plk1 targeting to the kinetochore and chromosome congression.

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.

Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells.

Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activities in a broad range of solid tumors. Here, we examined its effects on leukemic cells and found that, at 100 nM or less, it potently induced apoptosis in various leukemic cell lines and primary acute myeloid leukemia (AML) blasts. We then attempted to identify its mechanisms of action. Triptolide induced caspase-dependent cell death accompanied by a significant decrease in XIAP levels. Forced XIAP overexpression attenuated triptolide-induced cell death. Triptolide also decreased Mcl-1 but not Bcl-2 and Bcl-X(L) levels. Bcl-2 overexpression suppressed triptolide-induced apoptosis. Further, triptolide induced loss of the mitochondrial membrane potential and cytochrome C release. Caspase-9 knock-out cells were resistant, while caspase-8-deficient cells were sensitive to triptolide, suggesting criticality of the mitochondrial but not the death receptor pathway for triptolide-induced apoptosis. Triptolide also enhanced cell death induced by other anticancer agents. Collectively, our results demonstrate that triptolide decreases XIAP and potently induces caspase-dependent apoptosis in leukemic cells mediated through the mitochondrial pathway at low nanomolar concentrations. The potent antileukemic activity of triptolide in vitro warrants further investigation of this compound for the treatment of leukemias and other malignancies.

A novel mechanism of action of methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate: direct permeabilization of the inner mitochondrial membrane to inhibit electron transport and induce apoptosis.

Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO-Me) is a synthetic oleanolic acid derivative that displays antitumorigenic and anti-inflammatory activities, and we have previously reported that this agent potently activates the intrinsic apoptotic pathway in leukemia cells. In this study, we demonstrate that mitochondrial dysfunction induced by CDDO-Me is mediated by direct permeabilization of the inner mitochondrial membrane, which results in the rapid depletion of mitochondrial glutathione (GSXm), loss of cardiolipin, and inhibition of mitochondrial respiration. More importantly, we demonstrate that in addition to activating the intrinsic apoptotic pathway, the mitochondrial effects of CDDO-Me may mediate its anti-inflammatory activity by modulating the generation of superoxide anion (O2*). It is noteworthy that CDDO-Me did not increase the generation of O2* and pretreatment of leukemia cells with CDDO-Me prevented the increase of this reactive oxygen species elicited by inhibition of complex I or III in the absence of de novo protein synthesis. CDDO-Me, but not other inhibitors of respiration, induced a time- and dose-dependent, cyclosporin A-independent permeability transition (PT) of isolated mitochondria that was sensitive to sulfhydryl antioxidants but not to EDTA. PT induced by CDDO-Me and Ca2+ was accompanied by loss of GSXm, suggesting that the increased permeability of the inner mitochondrial membrane facilitates the loss of this antioxidant. Finally, transmission electron microscopy revealed that CDDO-Me rapidly induced caspase-independent mitochondrial swelling and loss of inner membrane structure before the release of cytochrome c. Taken together, our results indicate that CDDO-Me is a novel mitochondriotoxic agent that induces apoptosis and inhibits mitochondrial electron transport via perturbations in inner mitochondrial membrane integrity.

2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) directly targets mitochondrial glutathione to induce apoptosis in pancreatic cancer.

Surgical resection is the only curative strategy for pancreatic cancer (PC). Unfortunately, >80% of pancreatic cancer patients bear inoperable, locally advanced, chemoresistant tumors demonstrating the urgent need for development of novel therapeutic approaches to treat this disease. Here we report that the synthetic triterpenoid 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) antagonizes PC cell growth by inducing apoptosis at submicromolar concentrations. Notably, we demonstrate for the first time that the cytotoxicity of CDDO-Im is accompanied by the rapid and selective depletion of mitochondrial glutathione that results in accumulation of reactive oxygen species, oxidation of the cellular glutathione pool, loss of mitochondrial membrane potential, and phosphatidylserine externalization. The parent compound CDDO as well as the methyl ester of CDDO also depleted mitochondrial glutathione, demonstrating that this effect is mediated by the triterpenoid nucleus of these agents. Co-treatment with sulfhydryl nucleophiles completely prevented apoptosis and loss of viability induced by CDDO-Im, whereas alkylation of intracellular thiols by diethylmaleate or co-treatment with dithiothreitol decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in PC cells, suggesting that intracellular reduced thiols are functional targets of the electrophilic triterpenoid nucleus of CDDO and its derivatives. In conclusion, our report is the first to identify mitochondrial glutathione as a target of CDDO and its derivatives and demonstrates that depletion of this antioxidant in the mitochondria is an effective strategy to induce cell death in PC cells. These results suggest that CDDO and its derivatives may offer a clinical benefit for the treatment of PC.

Further psychometric evaluation and revision of the Mayo-Portland Adaptability Inventory in a national sample.

OBJECTIVES: To evaluate the internal consistency of the Mayo-Portland Adaptability Inventory (MPAI), further refine the instrument, and provide reference data based on a large, geographically diverse sample of persons with acquired brain injury (ABI). SUBJECTS: 386 persons, most with moderate to severe ABI. SETTINGS: Outpatient, community-based, and residential rehabilitation facilities for persons with ABI located in the United States: West, Midwest, and Southeast. METHODS: Rasch, item cluster, principal components, and traditional psychometric analyses for internal consistency of MPAI data and subscales. RESULTS: With rescoring of rating scales for 4 items, a 29-item version of the MPAI showed satisfactory internal consistency by Rasch (Person Reliability=.88; Item Reliability=.99) and traditional psychometric indicators (Cronbach's alpha=.89). Three rationally derived subscales for Ability, Activity, and Participation demonstrated psychometric properties that were equivalent to subscales derived empirically through item cluster and factor analyses. For the 3 subscales, Person Reliability ranged from.78 to.79; Item Reliability, from.98 to.99; and Cronbach's alpha, from.76 to.83. Subscales correlated moderately (Pearson r =.49-.65) with each other and strongly with the overall scale (Pearson r=.82-.86). CONCLUSIONS: Outcome after ABI is represented by the unitary dimension described by the MPAI. MPAI subscales further define regions of this dimension that may be useful for evaluation of clinical cases and program evaluation.

Nuclear localization is required for induction of apoptotic cell death by the Rb-associated p84N5 death domain protein.

The mechanisms utilized to transduce apoptotic signals that originate from within the nucleus, in response to DNA damage for example, are not well understood. Identifying these mechanisms is important for predicting how tumor cells will respond to genotoxic radiation or chemotherapy. The Rb tumor suppressor protein can inhibit apoptosis triggered by DNA damage, but how it does so is unclear. We have previously characterized a death domain protein, p84N5, that specifically associates with an amino-terminal domain of Rb protein. The p84N5 death domain is required for its ability to trigger apoptotic cell death. Association with Rb protein inhibits p84N5-induced apoptosis suggesting that it may be a mediator of Rb's effects on apoptosis. Unlike other death domain-containing apoptotic signaling proteins, however, p84N5 is localized predominantly within the nucleus of interphase cells. Here we test whether p84N5 requires nuclear localization in order to trigger apoptosis. We identify the p84N5 nuclear localization signal and demonstrate that nuclear localization is required for p84N5-induced apoptosis. To our knowledge, this identifies p84N5 as the first death-domain containing apoptotic signaling protein that functions within the nucleus. By analogy to other death domain containing proteins, p84N5 may play some role in apoptotic signaling within the nucleus. Further, p84N5 is a potential mediator of Rb protein's effects on DNA damage induced apoptosis.