Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

Analysis of differential gene expression in nontumorigenic glioblastoma multiforme

Loss of chromosome 10 represents the most common cytogenetic abnormality in high grade gliomas (glioblastoma multiforme). To identify genes involved in the malignant progression of human gliomas, a subtractive hybridization was performed between a tumorigenic glioblastoma cell line (LG11) and a nontumorgenic hybrid cell (LG11.3) containing an introduced chromosome 10. LG11 mRNA was subtracted from LG11.3 cDNA to produce cDNA probes enriched for sequences whose expression differs quantitatively from the parental tumorigenic cells. Both known and novel sequences were identified as a result of the subtraction. Northern blot analysis was then used to confirm differential expression of several subtracted clones. One novel clone, clone 17, identified a 2.6 kb message that showed a consistent two to four fold increase in expression in the LG11.3 nontumorigenic cells. Clone 17 (340 bp) was used successfully to screen for a near full-length version, RIG (regulated in glioma), which was 2,569 bp in size. The RIG cDNA sequence showed homology to clone 17 and to an anonymous EST (IB666), but to no previously identified genes. This screening effort also identified several independent clones representing novel sequences, most of which failed to show increased expression in the nontumorigenic GBM cells. Tissue distribution studies of RIG indicated highest levels of expression in human brain with appreciably lower levels in heart and lung. In vitro transcription and translation experiments demonstrated the ability of RIG to direct the synthesis of a 13 kD protein product. However, open reading frame analysis revealed no identify with previously described motifs or any known proteins. Using a combination of somatic cell hybrid panels and in situ hybridization, the RIG gene was mapped to chromosome 11p14-11p15. Further study of RIG and related gene products may provide insight into the negative regulation of glial oncogenesis. ^

Environmental and behavioral risk factors for moderate or severe rotavirus diarrhea in children less than 24 months

On a global basis rotaviruses are the most important agents involved in childhood diarrhea. In developing countries they account for 6% of all diarrheas and 20% of all diarrhea related deaths of children under 5 years of age, with over 1 billion episodes and over 4 million deaths annually. Given the disease burden, there is a need for better understanding the risk factors involved in rotavirus disease, to identify areas of intervention. In order to provide this information, two areas were developed: a review of the literature, examining the causal evidence for rotavirus diarrhea and a case comparison study. The case comparison study analyzed two areas: identifying climate factors and, identifying environmental and behavioral risk factors. The literature review showed that few analytical studies have identified specific risk factors such as home environment, and a winter seasonal trend for temperate areas, but in key areas evidence is contradictory. The case comparison study for climate factors demonstrated that seasonality occurs in a tropical country like Venezuela and that a complex interplay between weather conditions contribute to the seasonal pattern. A positive association between rain fall (OR 4.1); dew point (OR 2.3) and temperature differential during the day (OR 1.4) and, an inverse association with temperature (OR 0.5) and relative humidity (OR 0.8) was found. This information is useful in understanding the seasonal pattern of rotavirus and for planning health care needs. The second analysis demonstrated that environmental variables such as crowding (OR 14.3), contact with someone with an infectious disease (OR 4.9) and animal ownership (OR 2.3) were important. Restricting the analysis to animal owners demonstrated that living In a rural settling (OR 13.8), defecating in inappropriate places (OR 7.2), crowding(4.2) and indoor animals (4.0) are of importance. Behavioral variables identified were: lack of breast feeding (OR 4.0) and visiting when someone was sick (OR 3.4). Biological and demographic variables of importance were: age, with a dose response relationship; undernurishment (OR 11.3) and household per capita monthly income less than US $ 16.30 (OR 8.5). Using a diarrhea compeer group we found that, although some of the previous variables were of importance, no major differences were found. These findings are important in identifying paths for prevention and further research. ^

Issues related to HIV stigma: An embedded case study of PLHAS and community in Nepal

Little is known about HIV stigma in Nepal. Findings from other parts of the world suggest that HIV related stigma acts as a limiting factor for People Living with HIV & AIDS (PLHAs) from accessing needed health care and the subsequent psychological outcomes make PLHAs and their immediate family members more vulnerable. The purpose of this study is to understand and describe Nepalese PLHA's and community peoples' experiences regarding sources, types, causes and mechanism of HIV-associated stigma. This study aimed to best utilize the existing data. Study participants were recruited through local Community Based Organizations working in the field of HIV. The data, collected in the form of focus group discussions in workshop settings, were coded and analyzed. Study has revealed key issues related to HIV stigma in Nepal: hierarchy of stigma, denial and rejection, death as a form of punishment and Mumbaiya disease. A conceptual model was developed to explain the HIV stigma in Nepal. Our hope is that this finding, stigma being a contextual variable, will be helpful in triggering a novel public health discourse in the field of HIV stigma in Nepal, which will eventually be helpful for developing policies and interventions to eliminate HIV-associated stigma.^

Determining appropriate measurement methods for etiological research of computing-related musculoskeletal symptoms and disorders

Two studies among college students were conducted to evaluate appropriate measurement methods for etiological research on computing-related upper extremity musculoskeletal disorders (UEMSDs). ^ A cross-sectional study among 100 graduate students evaluated the utility of symptoms surveys (a VAS scale and 5-point Likert scale) compared with two UEMSD clinical classification systems (Gerr and Moore protocols). The two symptom measures were highly concordant (Lin's rho = 0.54; Spearman's r = 0.72); the two clinical protocols were moderately concordant (Cohen's kappa = 0.50). Sensitivity and specificity, endorsed by Youden's J statistic, did not reveal much agreement between the symptoms surveys and clinical examinations. It cannot be concluded self-report symptoms surveys can be used as surrogate for clinical examinations. ^ A pilot repeated measures study conducted among 30 undergraduate students evaluated computing exposure measurement methods. Key findings are: temporal variations in symptoms, the odds of experiencing symptoms increased with every hour of computer use (adjOR = 1.1, p < .10) and every stretch break taken (adjOR = 1.3, p < .10). When measuring posture using the Computer Use Checklist, a positive association with symptoms was observed (adjOR = 1.3, p < 0.10), while measuring posture using a modified Rapid Upper Limb Assessment produced unexpected and inconsistent associations. The findings were inconclusive in identifying an appropriate posture assessment or superior conceptualization of computer use exposure. ^ A cross-sectional study of 166 graduate students evaluated the comparability of graduate students to College Computing & Health surveys administered to undergraduate students. Fifty-five percent reported computing-related pain and functional limitations. Years of computer use in graduate school and number of years in school where weekly computer use was ≥ 10 hours were associated with pain within an hour of computing in logistic regression analyses. The findings are consistent with current literature on both undergraduate and graduate students. ^

The movement toward patient safety: State action related to reporting and disclosure of healthcare-associated infections

Increasing attention has been given to the problem of medical errors over the past decade. Included within that focused attention has been a strong interest in reducing the occurrence of healthcare-associated infections (HAIs). Acting concurrently with federal initiatives, the majority of U.S. states have statutorily required reporting and public disclosure of HAI data. Although the occurrence of these state statutory enactments and other state initiatives represent a recognition of the strong concern pertaining to HAIs, vast differences in each state’s HAI reporting and public disclosure requirements creates a varied and unequal response to what has become a national problem.^ The purpose of this research was to explore the variations in state HAI legal requirements and other state mandates. State actions, including statutory enactments, regulations, and other initiatives related to state reporting and public disclosure mechanisms were compared, discussed, and analyzed in an effort to illustrate the impact of the lack of uniformity as a public health concern.^ The HAI statutes, administrative requirements, and other mandates of each state and two U.S. territories were reviewed to answer the following seven research questions: How far has the state progressed in its HAI initiative? If the state has a HAI reporting requirement, is it mandatory or voluntary? What healthcare entities are subject to the reporting requirements? What data collection system is utilized? What measures are required to be reported? What is the public disclosure mechanism? How is the underlying reported information protected from public disclosure or other legal release?^ Secondary publicly available data, including state statutes, administrative rules, and other initiatives, were utilized to examine the current HAI-related legislative and administrative activity of the study subjects. The information was reviewed and analyzed to determine variations in HAI reporting and public disclosure laws. Particular attention was given to the seven key research questions.^ The research revealed that considerable progress has been achieved in state HAI initiatives since 2004. Despite this progress, however, when reviewing the state laws and HAI programs comparatively, considerable variations were found to exist with regards to the type of reporting requirements, healthcare facilities subject to the reporting laws, data collection systems utilized, reportable measures, public disclosure requirements, and confidentiality and privilege provisions. The wide variations in state statutes, administrative rules, and other agency directives create a fragmented and inconsistent approach to addressing the nationwide occurrence of HAIs in the U.S. healthcare system. ^

The prevention of catheter related bloodstream infections in a pediatric population

Catheter related bloodstream infections are a significant barrier to success in many inpatient healthcare facilities. The goal of this study was to analyze and determine if an evidence based methodology to reduce the number of catheter related bloodstream infections in a pediatric inpatient healthcare facility had significant impact on the infection rate. Catheter related bloodstream infection rates were compared before and after program implementation. The patient population was selected based upon a recommendation in the 2010 National Healthcare Safety Network report on device related infections. This report indicated a need for more data on pediatric populations requiring admission to a long term care facility. The study design is a retrospective cohort study. Catheter related bloodstream infection data was gathered between 2008 and 2011. In October of 2008 a program implementation began to reduce the number of catheter related bloodstream infections. The key components of this initiative were to implement a standardized catheter maintenance checklist, introduce the usage of a chlorhexadine gluconate based product for catheter maintenance and skin antisepsis, and a multidisciplinary education plan that focused on hand hygiene and aseptic technique. The catheter related bloodstream infection rate in 2008 was 21.21 infections per 1000 patient-line days. After program implementation the 2009 catheter related bloodstream infection rate dropped to 1.11 per 1000 patient-line days. The infection rates in 2010 and 2011 were 2.19 and 1.47 respectively. Additionally, this study demonstrated that there was a potential cost savings of $620,000 to $1,240,000 between 2008 and 2009. In conclusion, an evidence based program based upon CDC guidelines can have a significant impact on catheter related bloodstream infection rates. ^

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

Genetic analysis of Pitx2 in left -right asymmetry and cardiovascular development

Pitx2, a paired-related homeobox gene that is mutated in human Rieger Syndrome, plays a key role in transferring the early asymmetric signals to individual organs. Pitx2 encodes three isoforms, Pitx2a, Pitx2b and Pitx2c. I found that Pitx2c was the Pitx2 isoform for regulating left-right asymmetry in heart, lung and the predominant isoform in guts. Previous studies suggested that the generation of left-right asymmetry within individual organs is an all or none, random event. Phenotypic analysis of various Pitx2 allelic combinations, that encode graded levels of Pitx2c, reveals an organ-intrinsic mechanism for regulating left-right asymmetric morphogenesis based on differential response to Pitx2c levels. The heart needs low Pitx2c levels, while the lungs and duodenum require higher doses of Pitx2c. In addition, the duodenal rotation is under strict control of Pitx2c activity. Left-right asymmetry development for aortic arch arteries involves complex vascular remodeling. Left-sided expression of Pitx2c in these developing vessels implied its potential function in this process. In order to determine if Pitx2c also can regulate the left-right asymmetry of the aortic arch arteries, a Pitx2c-specific loss of function mutation is generated. Although in wild type mice, the direction of the aortic arch is always oriented toward the left side, the directions of the aortic arches in the mutants were randomized, showing that Pitx2c also determined the left-right asymmetry of these vessels. I have further showed that the cardiac neural crest wasn't involved in this vascular remodeling process. In addition, all mutant embryos had Double Outlet Right Ventricle (DORV), a common congenital heart disease. This study provided insight into the mechanism of Pitx2c-mediated late stages of left-right asymmetry development and identified the roles of Pitx2c in regulation of aortic arch remodeling and heart development. ^