Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Nitric oxide contributes to LPS/IFN-gamma-induced macrophage apoptosis via JNK and BCL-2 family regulation

Lipopolysaccharide (LPS) and interferon-gamma (IFN) activate macrophages and produce nitric oxide (NO) by initiating the expression of inducible Nitric Oxide Synthase (iNOS). Prolonged LPS/IFN-activation results in the death of macrophage-like RAW 264.7 cells and wild-type murine macrophages. This study was implemented to determine how NO contributes to LPS/IFN-induced macrophage death. The iNOS-specific inhibitor L-NIL protected RAW 264.7 cells from LPS/IFN-activated death, supporting a role for NO in the death of LPS/IFN-activated macrophages. A role for iNOS in cell death was confirmed in iNOS-/- macrophages which were resistant to LPS/IFN-induced death. Cell death was accompanied by nuclear condensation, caspase 3 activation, and PARP cleavage, all of which are hallmarks of apoptosis. The involvement of NO in modulating the stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) signal transduction pathway was examined as a possible mechanism of LPS/IFN-mediated apoptosis. Western analysis demonstrated that NO modifies the phosphorylation profile of JNK and promotes activation of JNK in the mitochondria in RAW 264.7 cells. Inhibition of JNK with sIRNA significantly reduced cell death in RAW 264.7 cells, indicating the participation of the JNK pathway in LPS/IFN-mediated death. JNK has been demonstrated to induce mitochondrial-mediated apoptosis through modulation of Bcl-2 family members. Therefore, the effect of NO on the balance between pro- and anti-apoptotic Bcl-2 family members was examined. In RAW 264.7 cells, Bim was upregulated and phosphorylated by LPS/IFN independently of NO. However, co-immunoprecipitation studies demonstrated that NO promotes the association of Bax with the BimL splice variant. Examination of Bax phosphorylation by metabolic labeling demonstrated that Bax is basally phosphorylated and becomes dephosphorylated upon LPS/IFN treatment. L-NIL inhibited the dephosphorylation of Bax, indicating that Bax dephosphorylation is NO-dependent. NO also mediated LPS/IFN-induced downregulation of Mcl-1, an anti-apoptotic Bcl-2 family member, as demonstrated by Western blotting for Mcl-1 protein expression. Thus, NO contributes to macrophage apoptosis via a JNK-mediated mechanism involving interaction between Bax and Bim, dephosphorylation of Bax, and downregulation of Mcl-1. ^

A systematic review and meta-analysis of diabetes disease management programs

Context. Despite the rapid growth of disease management programs, there are still questions about their efficacy and effectiveness for improving patient outcomes and their ability to reduce costs associated with chronic disease. ^ Objective. To determine the effectiveness of disease management programs on improving the results of HbA1c tests, lipid profiles and systolic blood pressure (SBP) readings among diabetics. These three quantitative measures are widely accepted methods of determining the quality of a patient's diabetes management and the potential for future complications. ^ Data Sources. MEDLINE and CINAHL were searched from 1950 to June 2008 using MeSH terms designed to capture all relevant studies. Scopus pearling and hand searching were also done. Only English language articles were selected. ^ Study Selection. Titles and abstracts for the 2347 articles were screened against predetermined inclusion and exclusion criteria, yielding 217 articles for full screening. After full article screening, 29 studies were selected for inclusion in the review. ^ Data Extraction. From the selected studies, data extraction included sample size, mean change over baseline, and standard deviation for each control and experimental arm. ^ Results. The pooled results show a mean HbA1c reduction of 0.64%, 95% CI (-0.83 to -0.44), mean SBP reduction of 7.39 mmHg (95% CI to -11.58 to -3.2), mean total cholesterol reduction of 5.74 mg/dL (95% CI, -10.01 to -1.43), and mean LDL cholesterol reduction of 3.74 mg/dL (95% CI, -8.34 to 0.87). Results for HbA1c, SBP and total cholesterol were statistically significant, while the results for LDL cholesterol were not. ^ Conclusions. The findings suggest that disease management programs utilizing five hallmarks of care can be effective at improving intermediate outcomes among diabetics. However, given the significant heterogeneity present, there may be fundamental differences with respect to study-specific interventions and populations that render them inappropriate for meta-analysis. ^

Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^

Can accreditation work as a tool for quality improvement in public health? A critical review

Public health departments play an important role in promoting and preserving the health of communities. The lack of a system to ensure their quality and accountability led to the development of a national voluntary accreditation program by Public Health Accreditation Board (PHAB). The concept that accreditation will lead to quality improvement in public health which will ultimately lead to healthy communities seems intuitive but lacks a robust body of evidence. A critical review of literature was conducted to explore if accreditation can lead to quality improvement in public health. The articles were selected from publically available databases using a specific set of criteria for inclusion, exclusion, and appraisal. To understand the relationship between accreditation and quality improvement, the potential strengths and limitations of accreditation process were evaluated. Recommendations for best practices are suggested so that public health accreditation can yield maximum benefits. A logic model framework to help depict the impact of accreditation on various levels of public health outcomes is also discussed in this thesis. The literature review shows that existing accreditation programs in other industries show limited but encouraging evidence that accreditation will improve quality and strengthen the delivery of public health services. While progress in introducing accreditation in public health can be informed by other accredited industries, the public health field has its own set of challenges. Providing incentives, creating financing strategies, and having a strong leadership will allow greater access to accreditation by all public health departments. The suggested recommendations include that continuous evaluation, public participation, systems approach, clear vision, and dynamic standards should become hallmarks of the accreditation process. Understanding the link between accreditation, quality improvement, and health outcomes will influence the successful adoption and implementation of the public health accreditation program. This review of literature suggests that accreditation is an important step in improving the quality of public health departments and in ultimately improving the health of communities. However, accreditation should be considered in an integrated system of tools and approaches to improve the public health practice. Hence, it is a means to an end - not an end unto itself.^

Understanding the roles of Non-homologous end joining and p53 after DNA damage

The inability to maintain genomic stability and control proliferation are hallmarks of many cancers, which become exacerbated in the presence of unrepaired DNA damage. Such genotoxic stresses trigger the p53 tumor suppressor network to activate transient cell cycle arrest allowing for DNA repair; if the damage is excessive or irreparable, apoptosis or cellular senescence is triggered. One of the major DNA repair pathway that mends DNA double strand breaks is non-homologous end joining (NHEJ). Abrogating the NHEJ pathway leads to an accumulation of DNA damage in the lymphoid system that triggers p53-mediated apoptosis; complete deletion of p53 in this system leads to aggressive lymphomagenesis. Therefore, to study the effect of p53-dependent cell cycle arrest, we utilized a hypomorphic, separation-of-function mutant, p53p/p, which completely abrogates apoptosis yet retains partial cell cycle arrest ability. We crossed DNA ligase IV deficiency, a downstream ligase crucial in mending breaks during NHEJ, into the p53p/p background (Lig4-/-p53p/p). The accumulation of DNA damage activated the p53/p21 axis to trigger cellular senescence in developing lymphoid cells, which absolutely suppressed tumorigenesis. Interestingly, these mice progressively succumb to severe diabetes. Mechanistic analysis revealed that spontaneous DNA damage accumulated in the pancreatic b-cells, a unique subset of endocrine cells solely responsible for insulin production to regulate glucose homeostasis. The genesis of adult b-cells predominantly occurs through self-replication, therefore modulating cellular proliferation is an essential component for renewal. The progressive accumulation of DNA damage, caused by Lig4-/-, activated p53/p21-dependent cellular senescence in mutant pancreatic b-cells that lead to islet involution. Insulin levels subsequently decreased, deregulating glucose homeostasis driving overt diabetes. Our Lig4-/-p53p/p model aptly depicts the dichotomous role of cellular senescence—in the lymphoid system prevents tumorigenesis yet in the endocrine system leads to the decrease of insulin-producing cells causing diabetes. To further delineate the function of NHEJ in pancreatic b-cells, we analyzed mice deficient in another component of the NHEJ pathway, Ku70. Although most notable for its role in DNA damage recognition and repair within the NHEJ pathway, Ku70 has NHEJ-independent functions in telomere maintenance, apoptosis, and transcriptional regulation/repression. To our surprise, Ku70-/-p53p/p mutant mice displayed a stark increase in b-cell proliferation, resulting in islet expansion, heightened insulin levels and hypoglycemia. Augmented b-cell proliferation was accompanied with the stabilization of the canonical Wnt pathway, responsible for this phenotype. Interestingly, the progressive onset of cellular senescence prevented islet tumorigenesis. This study highlights Ku70 as an important modulator in not only maintaining genomic stability through NHEJ-dependent functions, but also reveals a novel NHEJ-independent function through regulation of pancreatic b-cell proliferation. Taken in aggregate, these studies underscore the importance for NHEJ to maintain genomic stability in b-cells as well as introduces a novel regulator for pancreatic b-cell proliferation.

ROLE OF SOX9 IN UTERINE GLAND DEVELOPMENT AND DISEASE INITIATION

The female reproductive tract (FRT) develops midway through embryogenesis, and consists of oviducts, uterine horns, cervix and upper part of the vagina. The uterine horns are composed of an epithelial layer, luminal (LE) and glandular epithelium (GE), surrounded by a mesenchymal layer, the stroma and myometrium. Interestingly, in most mammals the GE forms after birth and it only becomes fully differentiated as the female reaches sexual maturity. Uterine glands (UG) are made up of GE and are present in all mammals. They secrete nutrients, cytokines and several other proteins, termed histotroph, that are necessary for embryo implantation and development. Experiments in ewes and mice have revealed that females who lack UGs are infertile mainly due to impaired implantation and early pregnancy loss, suggesting that UGs are essential for fertility. Fortunately for us, UGs develop after birth allowing us to peer into the genetic mechanism of tubulogenesis and branching morphogenesis; two processes that are disrupted in various adenocarcinomas (cancer derived from glands). We created 3D replicas of the epithelium lining the FRT using optical projection tomography and characterized UG development in mice using lineagetracing experiments. Our findings indicate that mouse UGs develop as simple tubular structures and later grow multiple secretory units that stem from the main duct. The main aim of this project was to study the role of SOX9 in the UGs. Preliminary studies revealed that Sox9 is mostly found in the nucleus of the GE. vii This observation led to the hypothesis that Sox9 plays a role in the formation and/or differentiation of the GE. To study the role of Sox9 in UGs differentiation, we conditionally knocked out and overexpressed Sox9 in both the LE and GE using the progesterone receptor (Pgr) promoter. Overexpressing Sox9 in the uterine epithelium, parts of the stroma, and myometrium led to formation of multiple cystic structures inside the endometrium. Histological analysis revealed that these structures appeared morphologically similar to structures present in histological tissue sections obtained from patients with endometrial polyps. We have accounted for the presence of simple and complex hyperplasia with atypia, metaplasia, thick-walled blood vessels, and stromal fibrosis; all “hallmarks��� that indicate overexpressing Sox9 leads to development of a polyp-like morphology. Therefore, we can propose the use of Sox9-cOE mice to study development of endometrial cystic lesions and disease progression into hyperplastic lesions.

The response of Texas public health agencies and workers to the emergency preparedness initiatives after 9/11

The purpose of this research was to better understand the impact of the terrorist attacks in 2001 on public health, particularly for Texas public health. This study employed mixed methods to examine changes to public health culture within Texas local public health agencies, important attitudes of public health workers toward responding to a disaster, and the funding policies that might ensure our investment in public health emergency preparedness is protected. ^ A qualitative analysis of interviews conducted with a large sample of public health officials in Texas found that all the constituent parts of a peculiar culture for public health preparedness existed that spanned the state's local health departments regardless of size, or funding level. The new preparedness culture in Texas had the hallmarks necessary for a robust public health preparedness and emergency response system. ^ The willingness of public health workers, necessary to make these kinds of changes and mount a disaster response was examined in one of Texas' most experienced disaster response teams—the public health workers for the City of Houston. A hypothesized latent variable model showed that willingness mediated all other factors in the model (self-efficacy, knowledge, barriers, and risk perception) for self-reported likelihood of reporting to work for a disaster. The RMSEA for the final model was 0.042 with a confidence interval of 0.036—0.049 and the chi-squared difference test was P=0.08, indicating a well-fitted model that suggests willingness is an important factor for consideration by preparedness planners and researchers alike. ^ Finally, with disasters on the rise and federal funding for preparedness dwindling, a review of states' policies for the distribution of these funds and their advantages and disadvantages were examined through a review of current literature and public documents, and a survey of state-level public health officials, emergency management professionals and researchers. Although the base plus per-capita method is the most common, it is not necessarily perceived to be the most effective. No clear "optimal" method emerged from the study, but recommendations for a strategic combination of three methods were made that has the potential to maximize the benefits of each method, while minimizing the weaknesses.^

Editoriall: A Holistic View of Family Preservation Services

One of the hallmarks of family preservation services is that they are holistic and attend to multiple dimensions of family life. In that spirit, this issue of the Family Preservation Journal provides a holistic view of these services, by offering research from the perspective of children, parents, caseworkers, and students of social work. These articles focus on the effectiveness of services, parents' perceptions of services, and the knowledge, attitudes and behavior of child welfare caseworkers. There should be something here for everyone who serves children and their families.

A study of nutrition promotion in and consumer nutrition environments of hospitals

Dietary intake is a complex, health-related behavior, and although individual-level theoretical models explain some variation in dietary intake, comprehensive theoretical models such as the ecological framework describe the multiple levels which influence diet-related behaviors. Thus, the ecological framework is a preferred model for designing comprehensive nutrition interventions. While ecological-based nutrition interventions have been described, little work has focused on interventions in the hospital setting. Because hospitals are considered the hallmarks of health, it might seem that hospitals would regularly engage in worksite nutrition promotion; however, recent publications and other anecdotal evidence have indicated otherwise. The first paper of this dissertation systematically reviewed the scientific literature between 1996 and 2012 and identified 13 outcome evaluation trials for hospital-based worksite nutrition interventions. Of these 13 interventions, only one intervention targeted three of the four levels of the ecological framework and no intervention targeted all four levels. Only half of the interventions targeted the physical environment of hospitals, thus warranting more investigation into this specific level of the ecological framework in this setting. ^ A critical type of nutrition-related physical environments is the consumer nutrition environment. Although other tools measure the consumer nutrition environments of stores and restaurants, no tool specifically measured the consumer nutrition environments of hospitals until the CDC developed the Healthy Hospital Environment Scan for Cafeterias, Vending Machines, and Gift Shops (HHES-CVG). The HHES-CVG, a tool which measures the consumer nutrition environments of hospital cafeterias, vending machines, and gifts shops, was released in November 2011, and in the second paper of this dissertation, the reliability of this tool was investigated. Two trained raters visited 39 hospitals across Southern California between February and May 2012, and based on analyses of the raters' findings, the HHES-CVG exhibited strong reliability metrics (inter-observer agreement between 74 and 100%, and an intraclass correlation coefficient of 0.961 for the overall nutrition composite score). Because the HHES-CVG was found to be a reliable tool, the third paper of this dissertation presented HHES-CVG results from the 39 hospitals. Overall, hospitals only scored about one-fourth of the total possible points for the nutrition composite score, indicating that most facilities do not have acceptable consumer nutrition environments. Some of the best practices observed in cafeterias were significantly associated with having a large facility and with having a contracted foodservice operation, but overall nutrition composite score was not associated with any specific facility or operation type. ^ The dissertation concluded that much work is needed in order to improve the consumer nutrition environments of hospitals. Practitioners and healthcare administrators should consider starting with ecological-based interventions addressing all levels including the physical environment.^

The functional role of the tumor suppressor MMAC /PTEN in glioblastoma multiforme and prostate cancer

Investigations into the molecular basis of glioblastoma multiforme led to the identification of a putative tumor suppressor gene, MMAC/ PTEN. Initial studies implicated MMAC/PTEN in many different tumor types, and identified a protein phosphatase motif in its sequence. This project aimed to identify the biological and biochemical functions of MMAC/PTEN by transiently expressing the gene in cancer cells that lack a functional gene product. ^ Expression of MMAC/PTEN mildly suppressed the growth of U251 human glioma cells and abrogated the growth advantage mediated by overexpression of the epidermal growth factor receptor (EGFR). Immunoblotting demonstrated that MMAC/PTEN expression did not affect the phosphorylation of the EGFR itself, or the intermediates of several downstream signaling pathways. However, MMAC/PTEN expression significantly reduced the phosphorylation and catalytic activity of the proto-oncogene Akt/PKB. While Akt/PKB regulates the survival of many cell types, expression of MMAC/PTEN did not induce apoptosis in adherent U251 cells. Instead, MMAC/PTEN expression sensitized the cells to apoptosis when maintained in suspension (anoikis). As the survival of suspended cells is one of the hallmarks leading to metastasis, MMAC/PTEN expression was examined in a system in which metastasis is more clinically relevant, prostate cancer. ^ Expression of MMAC/PTEN in both LNCaP and PC3-P human prostate cancer cells specifically inhibited Akt/PKB phosphorylation. MMAC/PTEN expression in LNCaP cells resulted in a profound inhibition of growth that was significantly greater than that achieved with expression of p53. Expression of MMAC/PTEN in PC3-P cells resulted in greater growth inhibition than was observed in U251 glioma cells, but less than was observed in LNCaP cells, or upon p53 expression. To determine if MMAC/PTEN could function as a tumor suppressor in vivo, the effects of MMAC/PTEN expression on PC3-P cells implanted orthotopically in nude mice were examined. The ex-vivo expression of MMAC/PTEN did not decrease tumor incidence, but it did significantly decrease tumor size and metastasis. In-vivo expression of MMAC/PTEN in pre-established PC3-P tumors did not significantly inhibit tumor incidence or size, but did inhibit metastasis formation. ^ These studies demonstrate that MMAC/PTEN is a novel and important tumor suppressor gene, which functions to downregulate an important cell survival signaling pathway. ^