Differential effects of complement activation products c3a and c5a on cardiovascular function in hypertensive pregnant rats.

Early-onset pre-eclampsia is characterized by decreased placental perfusion, new-onset hypertension, angiogenic imbalance, and endothelial dysfunction associated with excessive activation of the innate immune complement system. Although our previous studies demonstrated that inhibition of complement activation attenuates placental ischemia-induced hypertension using the rat reduced uterine perfusion pressure (RUPP) model, the important product(s) of complement activation has yet to be identified. We hypothesized that antagonism of receptors for complement activation products C3a and C5a would improve vascular function and attenuate RUPP hypertension. On gestational day (GD) 14, rats underwent sham surgery or vascular clip placement on ovarian arteries and abdominal aorta (RUPP). Rats were treated once daily with the C5a receptor antagonist (C5aRA), PMX51 (acetyl-F-[Orn-P-(D-Cha)-WR]), the C3a receptor antagonist (C3aRA), SB290157 (N(2)-[(2,2-diphenylethoxy)acetyl]-l-arginine), or vehicle from GD 14-18. Both the C3aRA and C5aRA attenuated placental ischemia-induced hypertension without affecting the decreased fetal weight or decreased concentration of free circulating vascular endothelial growth factor (VEGF) also present in this model. The C5aRA, but not the C3aRA, attenuated placental ischemia-induced increase in heart rate and impaired endothelial-dependent relaxation. The C3aRA abrogated the acute pressor response to C3a peptide injection, but it also unexpectedly attenuated the placental ischemia-induced increase in C3a, suggesting nonreceptor-mediated effects. Overall, these results indicate that both C3a and C5a are important products of complement activation that mediate the hypertension regardless of the reduction in free plasma VEGF. The mechanism by which C3a contributes to placental ischemia-induced hypertension appears to be distinct from that of C5a, and management of pregnancy-induced hypertension is likely to require a broad anti-inflammatory approach.

Molecular characterizations of Nop16 in murine mammary tumors with varying levels of c-Myc.

NOP16, also known as HSPC111, has been identified as a MYC and estrogen regulated gene in in vitro studies, hence coexpression levels were strongly correlated. Importantly, high expression of NOP16 was associated with poor clinical outcome in breast cancer patients. However, coexpression of NOP16, MYC and estrogen receptor (ESR1) varied widely in tumors and cell lines suggesting that transcriptional regulation differed according to pathological environments. The goal of this study was to determine the expression patterns of Nop16, Myc and Esr1 in murine mammary tumors with disparate histopathological and molecular features. We hypothesized that tumor environments with relatively high Myc levels would have different coexpression patterns than tumor environments with relatively low Myc levels. We measured levels of Myc and Nop16 mRNA and protein in tumors from WAP-c-myc mice that were of high grade and metastasized frequently. In contrast, Myc and Nop16 mRNA and proteins levels were significantly lower in the less aggressive tumors that developed in NRL-TGFα mice. Tumors from both mouse lines express ESR1 protein and we found that Esr1 mRNA levels correlated positively with Myc levels in both models. However, Myc and Nop16 transcript levels correlated positively only in tumors from NRL-TGFα mice. We identified prominent NOP16 protein in nuclei and less prominent staining in the cytoplasm of luminal cells of ducts and lobules from normal mammary glands as well as in hyperplasias and tumors obtained from NRL-TGFα mice. This staining pattern was reversed in tumors from WAP-c-Myc mice as nuclear staining was faint or absent and cytoplasmic staining more pronounced. In summary, the regulation of expression and localization of NOP16 varies in tumor environments with high versus low MYC levels and demonstrate the importance of stratifying clinical breast cancers based on MYC levels.

Critical Role for Macrophages in the Developmental Programming of Pancreatic ß-Cell Area in Offspring of Hypertensive Pregnancies.

Preeclampsia is a pregnancy-specific complication with long-term negative outcomes for offspring, including increased susceptibility to type 2 diabetes (T2D) in adulthood. In a rat reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia, maternal hypertension in conjunction with intrauterine growth restriction mimicked aspects of preeclampsia and resulted in female embryonic day 19 (e19) offspring with reduced ß-cell area and increased ß-cell apoptosis compared with offspring of sham pregnancies. Decreased pancreatic ß-cell area persisted to postnatal day 13 (PD13) in females and could influence whether T2D developed in adulthood. Macrophage changes also occurred in islets in T2D. Therefore, we hypothesized that macrophages are crucial to reduction in pancreatic ß-cell area in female offspring after chronic placental ischemia. Macrophage marker CD68 mRNA expression was significantly elevated in e19 and PD13 islets isolated from female RUPP offspring compared with sham. Postnatal injections of clodronate liposomes into female RUPP and sham offspring on PD2 and PD9 significantly depleted macrophages compared with injections of control liposomes. Depletion of macrophages rescued reduced ß-cell area and increased ß-cell proliferation and size in RUPP offspring. Our studies suggest that the presence of macrophages is important for reduced ß-cell area in female RUPP offspring and changes in macrophages could contribute to development of T2D in adulthood.

Arginase activity differs with allergen in the effector phase of ovalbumin- versus trimellitic anhydride-induced asthma.

Both trimellitic anhydride (TMA), a small molecular weight chemical, and ovalbumin (OVA), a reference protein allergen, cause asthma with eosinophilia. To test the hypothesis that different allergens elicit symptoms of asthma via different effector pathways, gene expression was compared in lungs of Balb/c mice sensitized with either TMA or OVA, followed by intratracheal challenge with TMA conjugated to mouse serum albumin (TMA-MSA) or OVA, respectively. Sensitized animals challenged with mouse serum albumin (MSA) alone were controls. Seventy-two hours after challenge, lung eosinophil peroxidase indicated that both allergens caused the same significant change in eosinophilia. Total RNA was isolated from lung lobes of 6-8 animals in each of four treatment groups and hybridized to Affymetrix U74Av2 GeneChips. False discovery rates (q-values) were calculated from an overall F test to identify candidate genes with differences in expression for the four groups. Using a q-value cutoff of 0.1, 853 probe sets had significantly different expression across the four treatment groups. Of these 853 probe sets, 376 genes had an Experimental/Control ratio of greater than 1.2 or less than 1/1.2 for either OVA- or TMA-treated animals, and 249 of the 376 genes were uniquely up- or down-regulated for OVA or TMA (i.e., differentially expressed with the allergen). qRT-PCR analysis of selected transcripts confirmed the gene expression analysis. Increases in both arginase transcript and enzyme activity were significantly greater in OVA-induced asthma compared to TMA-induced asthma. These data suggest that pathways of arginine metabolism and the importance of nitric oxide may differ in OVA- and TMA-induced asthma.

Improving rural cancer patients' outcomes: a group-randomized trial.

CONTEXT: Significant barriers exist in the delivery of state-of-the-art cancer care to rural populations. Rural providers' knowledge and practices, their rural health care delivery systems, and linkages to cancer specialists are not optimal; therefore, rural cancer patient outcomes are less than achievable. PURPOSE: To test the effects of a strategy targeting rural providers and their practice environment on patient travel for care, satisfaction, economic barriers, and health-related quality of life. METHODS: A group-randomized trial was conducted with 18 rural communities in the north-central United States. Twelve of these communities were included and defined as the unit of analysis for the patient outcomes portion of the study. The intervention targeted rural providers and their practice environment. The subjects were patients with breast, colorectal, lung, and prostate cancers from the rural communities. The main outcomes were patients' travel to obtain health care, satisfaction with care, perceptions of economic barriers to care, and health-related quality of life. In total, 881 patients were included. RESULTS: Group randomization was balanced. Travel for health care was significantly reduced in the community group exposed to the intervention during months 13 to 24 following cancer diagnosis. The mean miles traveled per patient were 1,326 (SE = 306) for the experimental group and 2,186 (SE = 347) for the control group (P = 0.03). No significant differences in satisfaction with care, economic barriers to care, or health-related quality of life were found. CONCLUSIONS: The intervention significantly reduced cancer patient travel for health care, which suggests that access to care improved in the experimental group. The results of this study do not allow conclusion that there was no effect on other patient outcomes. The results supported the study's conceptual framework and many of its hypotheses.

Short-term prophylactic tamoxifen reduces the incidence of antiestrogen-resistant/estrogen receptor-positive/progesterone receptor-negative mammary tumors.

Although many estrogen receptor-positive (ER+) breast cancers are effectively treated with selective estrogen receptor modulators and down-regulators (SERM/SERD), some are highly resistant. Resistance is more likely if primary cancers are devoid of progesterone receptors (PR-) or have high levels of growth factor activity. In this study, a transgenic mouse line that expresses transforming growth factor-alpha (NRL-TGFalpha mice) and that develops ER+/PR- mammary tumors was used to assess the possible effects of (a) therapeutic delivery of the SERM, tamoxifen, or SERD, ICI I82,780 (ICI), on the growth of established tumors and (b) short-term prophylactic tamoxifen administration on the initial development of new mammary tumors. To determine the therapeutic effects of tamoxifen and ICI on the growth of established tumors, mice were exposed to 3 weeks of drug treatment. Neither drug influenced tumor growth or glandular pathology. To determine if early prophylactic tamoxifen could alter tumorigenesis, a 60-day tamoxifen treatment was initiated in 8-week-old mice. Compared with placebo-treated mice, tamoxifen reduced tumor incidence by 50% and significantly decreased the degree of mammary hyperplasia. Prophylactic tamoxifen also significantly extended the life span of tumor-free mice. These data show that in this mouse model, established ER+/PR- mammary tumors are resistant to SERM/SERD treatment but the development of new mammary tumors can be prevented by an early course of tamoxifen. This study validates the utility of NRL-TGFalpha mice for (a) identifying candidate biomarkers of efficacious tamoxifen chemoprevention and (b) modeling the evolution of tamoxifen resistance.

Correlation of symptoms with vitamin D deficiency and symptom response to cholecalciferol treatment: a randomized controlled trial.

OBJECTIVE: To examine the association of symptoms with vitamin D deficiency and symptom response to cholecalciferol treatment in a randomized, double-blind, placebo-controlled trial. METHODS: Adult primary care patients in Duluth, Minnesota, were screened for vitamin D deficiency in February 2007. Participants completed questionnaires pertaining to a variety of symptoms, vitamin D intake, and selected medical conditions. Patients with mild to moderate vitamin D deficiency (25-hydroxyvitamin D [25(OH)D], 10-25 ng/mL) participated in a randomized controlled trial (RCT) of vitamin D replacement and its effect on symptoms. Participants were randomly assigned to receive 50 000 units of cholecalciferol (vitamin D3) weekly or placebo for 8 weeks. Patients with severe vitamin D deficiency (25[OH]D <10 ng/mL) were treated in an unblinded fashion, and symptoms were reevaluated post treatment. RESULTS: A total of 610 patients underwent initial screening, and 100 patients with mild to moderate vitamin D deficiency participated in the RCT. Thirty-eight severely deficient patients were treated in an unblinded fashion. On initial screening, 46.2% of participants were deficient in vitamin D. Self-reported vitamin D supplementation, milk intake, celiac disease, gastric bypass, and chronic pancreatitis were predictive of vitamin D status. Severely deficient participants reported increased musculoskeletal symptoms, depression (including seasonal), and higher (worse) scores on a fibromyalgia assessment questionnaire. In the RCT, the treated group showed significant improvement in fibromyalgia assessment scores (P = 0.03), whereas the placebo-treated participants did not. Severely deficient patients did not show symptom improvement over the 8-week trial period or when followed up 1 year later. CONCLUSIONS: Compared with participants in the placebo group, patients in the treatment group showed mild short-term improvement in the overall fibromyalgia impact score, but did not show significant improvement in most musculoskeletal symptoms or in activities of daily living.

Human influences on water quality in Great Lakes coastal wetlands.

A better understanding of relationships between human activities and water chemistry is needed to identify and manage sources of anthropogenic stress in Great Lakes coastal wetlands. The objective of the study described in this article was to characterize relationships between water chemistry and multiple classes of human activity (agriculture, population and development, point source pollution, and atmospheric deposition). We also evaluated the influence of geomorphology and biogeographic factors on stressor-water quality relationships. We collected water chemistry data from 98 coastal wetlands distributed along the United States shoreline of the Laurentian Great Lakes and GIS-based stressor data from the associated drainage basin to examine stressor-water quality relationships. The sampling captured broad ranges (1.5-2 orders of magnitude) in total phosphorus (TP), total nitrogen (TN), dissolved inorganic nitrogen (DIN), total suspended solids (TSS), chlorophyll a (Chl a), and chloride; concentrations were strongly correlated with stressor metrics. Hierarchical partitioning and all-subsets regression analyses were used to evaluate the independent influence of different stressor classes on water quality and to identify best predictive models. Results showed that all categories of stress influenced water quality and that the relative influence of different classes of disturbance varied among water quality parameters. Chloride exhibited the strongest relationships with stressors followed in order by TN, Chl a, TP, TSS, and DIN. In general, coarse scale classification of wetlands by morphology (three wetland classes: riverine, protected, open coastal) and biogeography (two ecoprovinces: Eastern Broadleaf Forest [EBF] and Laurentian Mixed Forest [LMF]) did not improve predictive models. This study provides strong evidence of the link between water chemistry and human stress in Great Lakes coastal wetlands and can be used to inform management efforts to improve water quality in Great Lakes coastal ecosystems.

Mechanisms of occupational asthma: Not all allergens are equal.

Asthma is a heterogeneous lung disorder characterized by airway obstruction, inflammation and eosinophil infiltration into the lung. Both genetics and environmental factors influence the expression of asthma, and not all asthma is the result of a specific immune response to allergen. Numerous asthma phenotypes have been described, including occupational asthma, and therapeutic strategies for asthma control are similar regardless of phenotype. We hypothesized that mechanistic pathways leading to asthma symptoms in the effector phase of the disorder differ with the inciting allergen. Since route of allergen exposure can influence mechanistic pathways, mice were sensitized by identical routes with a high molecular weight occupational allergen ovalbumin and a low molecular weight occupational allergen trimellitic anhydride (TMA). Different statistical methods with varying selection criteria resulted in identification of similar candidate genes. Array data are intended to provide candidate genes for hypothesis generation and further experimentation. Continued studies focused on genes showing minimal changes in the TMA-induced model but with clear up-regulation in the ovalbumin model. Two of these genes, arginase 1 and eotaxin 1 are the focus of continuing investigations in mouse models of asthma regarding differences in mechanistic pathways depending on the allergen. Microarray data from the ovalbumin and TMA model of asthma were also compared to previous data usingAspergillus as allergen to identify putative asthma 'signature genes', i.e. genes up-regulated with all 3 allergens. Array studies provide candidate genes to identify common mechanistic pathways in the effector phase, as well as mechanistic pathways unique to individual allergens.

Contributions of age and sex to heterogeneity of symptoms and effectiveness of secondary prevention strategies in asthma as modeled in the Guinea pig.

Previous studies in a guinea pig model of asthma have suggested that age and sex contribute both to the profile of asthma symptoms, i.e., asthma heterogeneity, as well as to the success of primary prevention strategies. The present study investigated the contributions of age and sex to the severity of central vs. peripheral airway hyperresponsiveness as well as to the effectiveness of secondary preventions strategies for asthma as modeled in the guinea pig. Experimental groups: Young/Young, sensitized and challenged before sexual maturity; Young/Adult, sensitized young and challenged after sexual maturity; Adult/Adult, sensitized and challenged after sexual maturity. Males and females were sensitized IP with 0.5 mg/kg ovalbumin (OVA) and challenged intratracheally with varying doses of OVA. Cellular infiltration into lung and lavage fluid, OVA specific IgG(1) as well as airway hyperresponsiveness to intravenous methacholine were determined 24 hr later. Airway hyperresponsiveness in central airways and peripheral lung was assessed by measurement of airway resistance, tissue damping and tissue elastance. Airway hyperresponsiveness with allergen sensitization and challenge was evident in male and female Adult/Adult animals and male Young/Young animals. Airway hyperresponsiveness in female Young/Young animals was not significant, despite marked airway eosinophilia. Changes in tissue elastance were more evident in OVA treated Adult/Adult compared to Young/Young animals. As allergen exposure decreased, a reduction in inflammation was seen in young females before other age sex groups. OVA induced increases in eosinophils were more pronounced in Young/Adult compared to Adult/Adult animals. Our results suggest that in asthmatic children, females may clinically benefit most from secondary prevention strategies to limit allergen exposure. In adult asthmatics, changes in tissue elastance may be significant, and secondary prevention strategies may be more effective in those sensitized as children compared to those sensitized as adults.

Integrated measures of anthropogenic stress in the U.S. Great lakes basin.

Integrated, quantitative expressions of anthropogenic stress over large geographic regions can be valuable tools in environmental research and management. Despite the fundamental appeal of a regional approach, development of regional stress measures remains one of the most important current challenges in environmental science. Using publicly available, pre-existing spatial datasets, we developed a geographic information system database of 86 variables related to five classes of anthropogenic stress in the U.S. Great Lakes basin: agriculture, atmospheric deposition, human population, land cover, and point source pollution. The original variables were quantified by a variety of data types over a broad range of spatial and classification resolutions. We summarized the original data for 762 watershed-based units that comprise the U.S. portion of the basin and then used principal components analysis to develop overall stress measures within each stress category. We developed a cumulative stress index by combining the first principal component from each of the five stress categories. Maps of the stress measures illustrate strong spatial patterns across the basin, with the greatest amount of stress occurring on the western shore of Lake Michigan, southwest Lake Erie, and southeastern Lake Ontario. We found strong relationships between the stress measures and characteristics of bird communities, fish communities, and water chemistry measurements from the coastal region. The stress measures are taken to represent the major threats to coastal ecosystems in the U.S. Great Lakes. Such regional-scale efforts are critical for understanding relationships between human disturbance and ecosystem response, and can be used to guide environmental decision-making at both regional and local scales.

Primary prevention of asthma: age and sex influence sensitivity to allergen-induced airway inflammation and contribute to asthma heterogeneity in Guinea pigs.

BACKGROUND: Limiting allergen exposure in the sensitization phase has been proposed as a means of primary prevention of asthma, but its effectiveness is debated. HYPOTHESIS: Primary prevention of asthma is more effective in limiting asthma symptoms in young guinea pigs compared with adults, whether males or females. METHODS: The following experimental groups were used: young/young, sensitized and challenged before sexual maturity; young/adult, sensitized young and challenged after sexual maturity; adult/adult, sensitized and challenged after sexual maturity. Males and females were sensitized intraperitoneally with varying doses of ovalbumin (OVA) and challenged intratracheally with a constant OVA dose. Cellular infiltration into lung and lavage fluid as well as airway hyperresponsiveness to intravenous methacholine was determined 24 h later. RESULTS: In unsensitized animals, density of resident inflammatory cells as well as baseline pulmonary function differed with age and sex. Maximum OVA-induced eosinophilia in females occurred at a lower sensitizing dose of OVA than in males, and the slopes of the dose-response relationship differed significantly between sexes. Young females had more pronounced increases in eosinophils compared with some adult treatment groups. The concentrations of OVA-specific antibodies were not directly related to differences in cellular infiltration. Airway hyperresponsiveness to methacholine challenge was observed in all treatment groups. CONCLUSION: Young animals require major reductions in allergen exposure compared with adults to effectively limit airway inflammation in primary prevention. Heterogeneity of asthma symptoms seen with age and sex suggests that primary prevention by limiting allergen exposure or treatment with anti-inflammatory or bronchodilator drugs may be more effective strategies for specific age and gender populations.

Environmentally stratified sampling design for the development of Great Lakes environmental indicators.

Understanding the relationship between human disturbance and ecological response is essential to the process of indicator development. For large-scale observational studies, sites should be selected across gradients of anthropogenic stress, but such gradients are often unknown for apopulation of sites prior to site selection. Stress data available from public sources can be used in a geographic information system (GIS) to partially characterize environmental conditions for large geographic areas without visiting the sites. We divided the U.S. Great Lakes coastal region into 762 units consisting of a shoreline reach and drainage-shed and then summarized over 200 environmental variables in seven categories for the units using a GIS. Redundancy within the categories of environmental variables was reduced using principal components analysis. Environmental strata were generated from cluster analysis using principal component scores as input. To protect against site selection bias, sites were selected in random order from clusters. The site selection process allowed us to exclude sites that were inaccessible and was shown to successfully distribute sites across the range of environmental variation in our GIS data. This design has broad applicability when the goal is to develop ecological indicators using observational data from large-scale surveys.

Lake Superior Rural Cancer Care Project, Part III: provider practice.

PURPOSE: Effective methods that encourage rural primary-care physicians to adopt state-of-the-art cancer-management practices are needed. The purpose of this study was to evaluate educational and systems strategies to improve rural primary-care physicians' cancer practice behaviors. DESCRIPTION OF STUDY: The Lake Superior Rural Cancer Care Project was a group-randomized, controlled trial conducted with 18 rural communities in the North Central United States over 4 years. Although the unit of analysis was the community, the subjects were 104 primary-care physicians and 2089 rural patients with cancer. The intervention was educational and comprised systems strategies that targeted rural primary-care physicians and their healthcare delivery systems. The outcome measures reported here were physician practice behaviors regarding cancer diagnosis, staging, treatment, clinical trial participation, and post-treatment surveillance. RESULTS: The intervention significantly improved 5 of the 37 cancer practice end points. The overall result of the study did not support the majority of the study hypotheses. Because 16 practice end points were found to be at acceptable performance levels, the possibility of a measurable intervention effect was limited. CLINICAL IMPLICATIONS: Earlier, the authors reported the results of the intervention on providers' cancer management knowledge, which showed significant improvement. The present study findings demonstrated that improving provider knowledge does not necessarily improve practice performance. Changing practice behaviors requires much more effort. Furthermore, interventions found to be effective in other diseases, types of providers, or settings may not work on rural providers for cancer management.