Cultural and linguistic disparities in dental utilisation in New South Wales, Australia.

OBJECTIVE: To examine the patterns and predictors of dental utilisation in culturally and linguistically diverse (CALD) and non-CALD groups in New South Wales. DESIGN: Secondary analysis of the 2013 and 2015 NSW Adult Population Health Survey (n=24,707). MAIN OUTCOME: Dental utilisation, defined as a dental visit within the last 12 months. CALD groups were defined using country of birth and language. Andersen's theoretical model was used. Chi-square test and multivariate logistic regression analysis adjusted for potential confounding. Sample weights adjusted for sampling design. RESULTS: Most (69%) of the population were Australian born; 20% spoke a language other than English at home. Dental utilisation was 58.9% and 63.9% for CALD and non-CALD groups respectively. The foreign-born non-English speaking group had the highest level of education (60%) but lower levels of dental utilisation (OR:0.81, CI 0.69-0.94) than all groups. Australian born non-English speakers had similar levels of dental utilisation to the reference group (OR:1.27, CI 0.99-1.63). CONCLUSION: There are significant disparities in dental care utilisation among CALD populations. Foreign born, non-English speaking CALD migrants, and people experiencing socioeconomic disadvantage, are at greatest risk of inadequate dental utilisation. Furthermore, the combination of predisposing factors, language and cultural barriers compound disparities in oral health care utilisation. This data highlights the need for oral healthcare services that are sensitive to population needs, to reduce disparities among CALD communities residing in NSW.

Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma.

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)-ß-mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single-cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

Indigenous cultural competence: A dental faculty curriculum review.

BACKGROUND: Indigenous Australians have more than double the rate of poor oral health than their non-Indigenous counterparts. Cultural competence of dental and oral health practitioners is fundamental to health care and quality of life in addressing health disparities in minority cultural groups in Australia. Higher education curricula reviews have identified the need for institutions to incorporate Indigenous culture and knowledge more widely into the curricula to improve educational outcomes for Indigenous Australians and to increase cultural competence for all students. AIM: The aim of this research was to provide a baseline analysis of Indigenous cultural competence curricula practices to ascertain changes required within Faculty of Dentistry programmes at the University of Sydney to enable students to become more culturally competent upon graduation. METHODS: Staff and students of the Doctor of Dental Medicine and Bachelor of Oral Health programmes at the Faculty of Dentistry, University of Sydney participated in an online survey. Quantitative analysis of the survey data was conducted using integrated research electronic data capture survey tools, with open-ended questions being coded to common responses for those questions. RESULTS: A total of 69 staff (71%) and 191 students (51%) participated in the online survey. The majority of participants perceived there was limited Indigenous content in the curriculum. Most participants reported that Indigenous curriculum was integrated into several units of study. The main pedagogical method for curriculum delivery was lectures, followed by case studies and group discussions. CONCLUSION: Although some Indigenous content exists in dental faculty curriculum, in-depth investigation is required to develop a comprehensive, evidenced-based Indigenous cultural competence teaching framework, for integration into Doctor of Dental Medicine and Bachelor of Oral Health curricula.

Development of life story experience (LSE) scales for migrant dentists in Australia: a sequential qualitative-quantitative study.

INTRODUCTION: The integration of qualitative and quantitative approaches introduces new avenues to bridge strengths, and address weaknesses of both methods. OBJECTIVE: To develop measure(s) for migrant dentist experiences in Australia through a mixed methods approach. METHODS: The sequential qualitative-quantitative design involved first the harvesting of data items from qualitative study, followed by a national survey of migrant dentists in Australia. Statements representing unique experiences in migrant dentists' life stories were deployed the survey questionnaire, using a five-point Likert scale. Factor analysis was used to examine component factors. RESULTS: Eighty-two statements from 51 participants were harvested from the qualitative analysis. A total of 1,022 of 1,977 migrant dentists (response rate 54.5%) returned completed questionnaires. Factor analysis supported an initial eight-factor solution; further scale development and reliability analysis led to five scales with a final list of 38 life story experience (LSE) items. Three scales were based on home country events: health system and general lifestyle concerns (LSE1; 10 items), society and culture (LSE4; 4 items) and career development (LSE5; 4 items). Two scales included migrant experiences in Australia: appreciation towards Australian way of life (LSE2; 13 items) and settlement concerns (LSE3; 7 items). CONCLUSION: The five life story experience scales provided necessary conceptual clarity and empirical grounding to explore migrant dentist experiences in Australia. Being based on original migrant dentist narrations, these scales have the potential to offer in-depth insights for policy makers and support future research on dentist migration.

Cell migration in paediatric glioma; characterisation and potential therapeutic targeting.

BACKGROUND: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. METHODS: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. RESULTS: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. CONCLUSIONS: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Methylation-specific multiplex ligation-dependent probe amplification identifies promoter methylation events associated with survival in glioblastoma.

DNA methylation plays an important role in cancer biology and methylation events are important prognostic and predictive markers in many tumor types. We have used methylation-specific multiplex ligation-dependent probe amplification to survey the methylation status of MGMT and 25 tumor suppressor genes in 73 glioblastoma cases. The data obtained was correlated with overall survival and response to treatment. The study revealed that methylation of promoter regions in TP73 (seven patients), THBS1 (eight patients) and PYCARD (nine patients) was associated with improved outcome, whereas GATA5 (21 patients) and WT1 (24 patients) promoter methylation were associated with poor outcome. In patients treated with temozolomide and radiation MGMT and PYCARD promoter methylation events remained associated with improved survival whereas GATA5 was associated with a poor outcome. The identification of GATA5 promoter methylation in glioblastoma has not previously been reported. Furthermore, a cumulative methylation score separated patients into survival groups better than any single methylation event. In conclusion, we have identified specific methylation events associated with patient outcome and treatment response in glioblastoma, and these may be of functional and predictive/prognostic significance. This study therefore provides novel candidates and approaches for future prospective validation.

A Randomised Phase II Trial of Hippocampal Sparing Versus Conventional Whole Brain Radiotherapy After Surgical Resection or Radiosurgery in Favourable Prognosis Patients With 1-10 Brain Metastases.

AIMS: To assess in patients with 1-10 brain metastases, each of which has been treated by neurosurgery or stereotactic radiosurgery, whether hippocampal sparing whole brain radiotherapy (HS-WBRT) better spares neurocognitive function (NCF) than standard WBRT. Further, to assess whether a phase III randomised trial of HS-WBRT would be feasible in the UK. MATERIALS AND METHODS: A multicentre, randomised, open label phase II trial was undertaken, randomising patients to 30Gy in 10 fractions of WBRT or HS-WBRT. The primary endpoint was decline in Total recall using Hopkins Verbal Learning Test Revised (HVLT-R) at 4 months post treatment. To assess this, we aimed to recruit 84 patients over 3 years. Secondary endpoints included further measures of NCF, quality of life, duration of functional independence, local control of treated metastases, development of new metastases, disease control within the hippocampal regions, overall survival, steroid and antiepileptic medication requirements, and toxicity. RESULTS: The trial closed prematurely due to slower than anticipated recruitment. From April 2016 to January 2018, 23 patients were randomised. Follow up was a median of 25 months. Fifteen patients (6 WBRT, 9 HS-WBRT) were assessed for the primary endpoint; of these, 1 in each arm experienced significant decline in the 4-month HVLT-R Total recall score (p = 0.8). Patients in the HS-WBRT arm experienced less insomnia (p < 0.01) and drowsiness (p < 0.01). There were no differences in other secondary endpoints. CONCLUSION: A phase III randomised trial of HS-WBRT was shown not to be feasible at this time in the UK. As most randomised trials of HS-WBRT reported to date share common endpoints, including NCF, an individual patient data meta-analysis should be undertaken.

Intergenerational effects of racism on amygdala and hippocampus resting state functional connectivity.

Racism is an insidious problem with far-reaching effects on the lives of Black, Indigenous, and People of Color (BIPOC). The pervasive negative impact of racism on mental health is well documented. However, less is known about the potential downstream impacts of maternal experiences of racism on offspring neurodevelopment. This study sought to examine evidence for a biological pathway of intergenerational transmission of racism-related trauma. This study examined the effects of self-reported maternal experiences of racism on resting state functional connectivity (rsFC) in n = 25 neonates (13 female, 12 male) birthed by BIPOC mothers. Amygdala and hippocampus are brain regions involved in fear, memory, and anxiety, and are central nodes in brain networks associated with trauma-related change. We used average scores on the Experiences of Racism Scale as a continuous, voxel-wise regressor in seed-based, whole-brain connectivity analysis of anatomically defined amygdala and hippocampus seed regions of interest. All analyses controlled for infant sex and gestational age at the 2-week scanning session. More maternal racism-related experiences were associated with (1) stronger right amygdala rsFC with visual cortex and thalamus; and (2) stronger hippocampus rsFC with visual cortex and a temporo-parietal network, in neonates. The results of this research have implications for understanding how maternal experiences of racism may alter neurodevelopment, and for related social policy.

Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

ATR-dependent radiation-induced gamma H2AX foci in bystander primary human astrocytes and glioma cells.

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.

Moving from advocacy to activism? The fourth WHO global forum on human resources for health and implications for dentistry.

As we debate shaping the future oral health workforce within the UK, to meet the needs of current and future populations, it is helpful to take an international perspective on this very important issue. Globally, there is a strong recognition that human resources for health (HRH) are fundamentally important to deliver effective care, accessible to all people. This paper reviews the outcome of the fourth global forum held by the World Health Organisation (WHO) in Dublin which highlighted the urgency for action. The main objectives of the forum were to advance the implementation of (i) the WHO Global Strategy on HRH 2030 and (ii) the United Nations High-Level Commission's Health Employment and Economic Growth recommendations. From an oral health perspective, the global burden of oral disease remains huge with untreated dental caries, periodontal disease and tooth loss ranking among the most prevalent conditions worldwide. Major considerations are how dental education, practice delivery and/or oral health systems as a whole could and should innovate to accommodate the growing needs of the population. As dental professionals, it also becomes necessary for us to engage and play a proactive role in this change process. Due to growing differences between population needs and available services, it is necessary for oral health personnel to work more closely with the broader health workforce so as to identify solutions that are in the best interests of the patients and populations at large.

Dose- and time-dependent changes in gene expression in human glioma cells after low radiation doses.

We have used DNA microarrays to identify changes in gene expression in cells of the radioresistant human glioma cell lines T98G and U373 after low radiation doses (0.2-2 Gy). Using Bayesian linear models, we have identified a set of genes that respond to low doses of radiation; furthermore, a hypothesis-driven approach to data analysis has allowed us to identify groups of genes with defined non-linear dose responses. Specifically, one of the cell lines we have examined (T98G) shows increased radiosensitivity at low doses (low-dose hyper-radiosensitivity, HRS); thus we have also assessed sets of genes whose dose response mirrors this survival pattern. We have also investigated a time course for induction of genes over the period when the DNA damage response is expected to occur. We have validated these data using quantitative PCR and also compared genes up-regulated in array data to genes present in the polysomal RNA fraction after irradiation. Several of the radioresponsive genes that we describe code for proteins that may have an impact on the outcome of irradiation in these cells, including RAS homologues and kinases involved in checkpoint signaling, so understanding their differential regulation may suggest new ways of altering radioresistance. From a clinical perspective these data may also suggest novel targets that are specifically up-regulated in gliomas during radiotherapy treatments.

Identification of a silencer module which selectively represses cyclic AMP-responsive element-dependent gene expression.

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.

Integrin-mediated signaling events in human endothelial cells.

Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. In the present study, we investigate whether integrin-mediated anchorage to a substratum coated with the extracellular matrix protein fibronectin regulates growth factor signaling events in human endothelial cells. We show that cell adhesion to fibronectin and growth factor stimulation trigger distinct initial tyrosine phosphorylation events in endothelial cells. Thus, integrin-dependent adhesion of endothelial cells leads to tyrosine phosphorylation of both focal adhesion kinase and paxillin, but not of several growth factor receptors. Conversely, EGF stimulation causes receptor autophosphorylation, with no effect on focal adhesion kinase or paxillin tyrosine phosphorylation. Adhesion to fibronectin, in the absence of growth factors, leads to activation of MAPK. In addition, adhesion to fibronectin also potentiates growth factor signaling to MAPK. Thus, polypeptide growth factor activation of MAPK in anchored cells is far more effective than in cells maintained in suspension. Other agonists known to activate MAPK were also examined for their ability to activate MAPK in an anchorage-dependent manner. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor alpha, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin-adherent cells than in suspended cells. In addition, tumor necrosis factor alpha activation of c-Jun kinase (JNK) was also much more robust in anchored cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression.

Simultaneous 68Ga DOTATATE Positron Emission Tomography/Magnetic Resonance Imaging in Meningioma Target Contouring: Feasibility and Impact Upon Interobserver Variability Versus Positron Emission Tomography/Computed Tomography and Computed Tomography/Magnetic Resonance Imaging.

AIMS: The increasing use of highly conformal radiation techniques to treat meningioma confers a greater need for accurate targeting. Several groups have shown that positron emission tomography/computed tomography (PET/CT) information alters meningioma targets contoured by single observers, but whether this translates into improved accuracy has not been defined. As magnetic resonance imaging (MRI) is the cornerstone of meningioma target contouring, simultaneous PET/MRI may be superior to PET/CT. We assessed whether 68Ga DOTATATE PET imaging (from PET/CT and PET/MRI) reduced interobserver variability (IOV) in meningioma target volume contouring. MATERIALS AND METHODS: Ten patients with meningioma underwent simultaneous 68Ga DOTATATE PET/MRI followed by PET/CT. They were selected as it was anticipated that target volume definition in their cases would be particularly challenging. Three radiation oncologists contoured target volumes according to an agreed protocol: gross tumour volume (GTV) and clinical target volume (CTV) on CT/MRI alone, CT/MRI+PET(CT) and CT/MRI+PET(MRI). GTV/CTV Kouwenhoven conformity levels (KCL), regions of contour variation and qualitative differences between PET(CT) and PET(MRI) were evaluated. RESULTS: There was substantial IOV in contouring. GTV mean KCL: CT/MRI 0.34, CT/MRI+PET(CT) 0.38, CT/MRI+PET(MRI) 0.39 (P = 0.06). CTV mean KCL: CT/MRI 0.31, CT/MRI+PET(CT) 0.35, CT/MRI+PET(MRI) 0.35 (P = 0.04 for all groups; P > 0.05 for individual pairs). One observer consistently contoured largest and one smallest. Observers rarely decreased volumes in relation to PET. Most IOV occurred in bone followed by dural tail, postoperative bed and venous sinuses. Tumour edges were qualitatively clearer on PET(MRI) versus PET(CT), but this did not affect contouring. CONCLUSION: IOV in contouring challenging meningioma cases was large and only slightly improved with the addition of 68Ga DOTATATE PET. Simultaneous PET/MRI for meningioma contouring is feasible, but did not improve IOV versus PET/CT. Whether volumes can be safely reduced according to PET requires evaluation.

New vaccine administration practice recommendations from the Canadian Immunization Guide.

BACKGROUND: The Canadian Immunization Guide (CIG) is published online by the Public Health Agency of Canada and summarizes guidance on vaccines for human use into a single resource. Chapters are reviewed and updated on a regular basis. Vaccine administration is a critical part of any immunization program. Recently, the CIG chapter on vaccine administration practices was updated. OBJECTIVE: To provide highlights of recent changes to the Vaccine Administration Practices chapter of the CIG. APPROACH: Vaccine-specific guidance in the CIG is based on National Committee on Immunization (NACI) and Committee to Advise on Tropical Medicine and Travel (CATMAT) recommendations as well as new recommendations developed by the CIG Working Group members and NACI Secretariat technical staff. New recommendations are based on a review of the literature, including systematic reviews when available, a review of guidance provided by other National Immunization Technical Advisory Groups and expert opinion. The revisions are approved by the Working Group chair, as well as NACI. RESULTS: Highlights of new recommendations include the following: vaccine providers should adhere to jurisdictional or organizational policies and procedures regarding combining the contents of multi-dose vials; clinical judgement should be used when selecting needle length for intramuscular injections that takes into account the vaccine recipient's weight, gender and age; filter needles are not recommended for vaccine administration as they may filter out active ingredients such as adjuvants; an injection site other than in an area where lymphatic drainage may be impaired should be considered; there is no evidence or theoretical rationale for avoiding injection through a tattoo or superficial birthmark; and immunization pain management strategies have now been developed for all ages. CONCLUSION: Recommendations in vaccine administration practices have recently been changed in some important ways. The Public Health Agency of Canada is committed to providing information on immunization in an easily accessible, reader-friendly format for healthcare providers and policy-makers.

Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer.

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.