Laser-engineered dissolving microneedle arrays for protein delivery: potential for enhanced intradermal vaccination

OBJECTIVES: We aimed to highlight the utility of novel dissolving microneedle (MN)-based delivery systems for enhanced transdermal protein delivery. Vaccination remains the most accepted and effective approach in offering protection from infectious diseases. In recent years, much interest has focused on the possibility of using minimally invasive MN technologies to replace conventional hypodermic vaccine injections.

METHODS: The focus of this study was exploitation of dissolving MN array devices fabricated from 20% w/w poly(methyl vinyl ether/maleic acid) using a micromoulding technique, for the facilitated delivery of a model antigen, ovalbumin (OVA).

KEY FINDINGS: A series of in-vitro and in-vivo experiments were designed to demonstrate that MN arrays loaded with OVA penetrated the stratum corneum and delivered their payload systemically. The latter was evidenced by the activation of both humoral and cellular inflammatory responses in mice, indicated by the production of immunoglobulins (IgG, IgG1, IgG2a) and inflammatory cytokines, specifically interferon-gamma and interleukin-4. Importantly, the structural integrity of the OVA following incorporation into the MN arrays was maintained.

CONCLUSION: While enhanced manufacturing strategies are required to improve delivery efficiency and reduce waste, dissolving MN are a promising candidate for 'reduced-risk' vaccination and protein delivery strategies.

Correlates of food choice in unemployed young people: The role of demographic factors, self-efficacy, food involvement, food poverty and physical activity

Associations between socio-demographic and psychological factors and food choice patterns were explored in unemployed young people who constitute a vulnerable group at risk of poor dietary health. Volunteers (N = 168), male (n = 97) and female (n = 71), aged 15–25 years were recruited through United Kingdom (UK) community-based organisations serving young people not in education training or employment (NEET). Survey questionnaire enquired on food poverty, physical activity and measured responses to the Food Involvement Scale (FIS), Food Self-Efficacy Scale (FSS) and a 19-item Food Frequency Questionnaire (FFQ). A path analysis was undertaken to explore associations between age, gender, food poverty, age at leaving school, food self-efficacy (FS-E), food involvement (FI) (kitchen; uninvolved; enjoyment), physical activity and the four food choice patterns (junk food; healthy; fast food; high fat). FS-E was strong in the model and increased with age. FS-E was positively associated with more
frequent choice of healthy food and less frequent junk or high fat food (having controlled for age, gender and age at leaving school). FI (kitchen and enjoyment) increased with age. Higher FI (kitchen) was associated with less frequent junk food and fast food choice. Being uninvolved with food was associated with
more frequent fast food choice. Those who left school after the age of 16 years reported more frequent physical activity. Of the indirect effects, younger individuals had lower FI (kitchen) which led to frequent junk and fast food choice. Females who were older had higher FI (enjoyment) which led to less frequent fast food choice. Those who had left school before the age of 16 had low food involvement (uninvolved) which led to frequent junk food choice. Multiple indices implied that data were a good fit to the model which indicated a need to enhance food self-efficacy and encourage food involvement in order to improve dietary health among these disadvantaged young people.

Tumorigenesis and peritoneal colonization from fallopian tube epithelium

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system.

Nong Thale Pron - a key site from southern Thailand for studying monsoon variability during the past 15000 years

Studies of marine sediments, cave speleothemes, annually laminated corals, and tree rings from Asian monsoon regions have added knowledge to our understanding of the factors that control inter-annual to millennial monsoon variability in the past and have provided important constraints for climate modeling scenarios. In contrast, the spatial and temporal pattern of sub-millennial scale monsoon variability and its impact on land cover in SE Asia are still unresolved. This shortcoming stems from the fact that temporally well-resolved paleo-environmental studies are missing from large parts of SE Asia, especially from Thailand. Given that global and regional climate models are increasingly using terrestrial paleo- data to test their performance, past changes in land cover are therefore important variables to better understand feedbacks between different Earth systems. We obtained sediments from Lake Nong Thale Pron, in southern Thailand (8º 10`N, 99 º23`E; 380 m.asl). The aim of our study is to reconstruct lake status changes and to evaluate whether the extent of these changes are linked to known shifts in monsoon intensity and variability. Preliminary results show that lake infilling started more than 15,000 years ago and that the sediments cover the last deglaciation and the Holocene. Current analyses include Itrax XRF core scanning, loss-on-ignition (LOI at 950 and 550ºC), CN elemental and isotopic composition. We expect that our results will be able to give a picture of how the lake's status has changed over time and whether the extent of these changes is linked to known shifts in monsoon intensity and variability.

Electrochemical promotion of a Pt catalyst supported on La 0.6Sr 0.4Co 0.2Fe 0.8O 3 - δ hollow fibre membranes

The use of wireless electrochemical promotion of catalysis (EPOC) of a Pt catalyst supported on a mixed ionic electronic conducting hollow fibre membranes is investigated. This reactor configuration offers high surface areas per unit volume and is ideally suited for scaled-up applications. The MIEC membrane used is the La _0.6Sr _0.4Co _0.2Fe _0.8O ₃ perovskite (LSCF) with a Pt catalyst film deposited on the outer surface of the LSCF membrane. Experimental results showed that after initial catalyst deactivation (in the absence of an oxygen chemical potential difference across the membrane) the catalytic rate can be enhanced by using an oxygen sweep and wireless EPOC can be used for the in situ regeneration of a deactivated catalyst. © 2012 Elsevier B.V.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.

MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.

RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.

CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

The role of secreted frizzled-related protein 2 expression in prostate cancer

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2.

METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients.

CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

The HIF-1alpha C1772T polymorphism may be associated with susceptibility to clinically localised prostate cancer but not with elevated expression of hypoxic biomarkers

We investigated the role of the C1772T polymorphisms in exon 12 of the Hypoxia-inducible factor-1 alpha (HIF-1alpha) gene C1772T genotype in prostate cancer (PCa) and amplification of the hypoxic response. We identified the heterozygous germline CT genotype as an increased risk factor for clinically localised prostate cancer (Odds ratio = 6.2; p < 0.0001). While immunostaining intensity for HIF-1alpha and VEGF was significantly enhanced in 75% of PCa specimens when compared to matched benign specimens (p < 0.0001), the CT genotype did not modulate the kinetics of HIF-1alpha protein expression in hypoxia in vitro, and was not associated with enhanced expression of hypoxic biomarkers. This study provides the first evidence of an increased risk for clinically localised prostate cancer in men carrying the C1772T HIF-1alpha gene polymorphism. Although our results did not suggest an association between expression of hypoxic biomarkers and genotype status, the correlation may merit further investigation.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.