Stem Cell and Biological Interventions to treat Allergic Airway Disease

The aim of this work was to investigate immune modulation with a particular focus on airway inflammation and allergic pathogenesis. This was probed in a model of pathogen driven immunomodulation (B. pertussis), and two models of therapeutic intervention namely immunisation (attenuated B. pertussis, BPZE1) or using a candidate cell therapy approach (mesenchymal stem cells, MSC). This work demonstrated that, in contrast to virulent B. pertussis, an attenuated, candidate vaccine strain of B. pertussis, BPZE1, did not enhance but rather reduced allergendriven airway pathology supporting findings that suggest allergic asthma is linked not just to a CD4+ T cell profile, but also to the degree of airway damage at the time of priming. The second approach sought to ascertain whether a candidate stem cell therapy could modulate immunity in vivo to a degree that was sufficient to suppress allergic lung inflammation. The work presented here demonstrated that adult bone marrow-derived allogeneic MSC actively prevent the induction of allergen driven pathology in a murine model. Prior stimulation of MSC with IFN-γ increased their protective effect. An increase in IL-10 as a result of MSC delivery suggested a role for MSC in Treg induction. The immune mechanisms by which MSC confer protection in this model were probed and demonstrated that MSC generate the expansion of Treg subsets, CD4+FoxP3 and CD8+ γ/δ T cells, in the lung. Moreover, expansion of Treg was proven to exert a functional effect as depletion of these cells resulted in a negation of the protective effect of MSC. In an experimental asthma model, where mice devoid of naturally occurring Treg, delivery of MSC fails to impair the development of allergic airway pathology and class switching to IgE. A reduction in airway eosinophilia in the absence of regulatory T cells suggested an alternative mechanism of protection employed by MSC. Pre-incubation with MSC inhibited the expression of important adhesion markers on eosinophils which most likely affected their ability to migrate as demonstrated by the inhibitory effect on their chemotactic migration. These data suggests a critical role for soluble factor secretion by MSC contributing to the reduction of airway eosinophilia. Collectively, these data provides a fundamental insight into novel therapeutic approaches that can profoundly influence the allergic airway response

Host chemokine signature as a biomarker for the detection of pre-cancerous cervical lesions

Background The ability to distinguish which hrHPV infections predispose to significant disease is ever more pressing as a result of the increasing move to hrHPV testing for primary cervical screening. A risk-stratifier or “triage” of infection should ideally be objective and suitable for automation given the scale of screening. Results CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL12 emerged as the strongest, candidate biomarkers to detect underlying disease [cervical intraepithelial neoplasia grade 2 or worse (CIN2+)]. For CIN2+, CCL2 had the highest area under the curve (AUC) of 0.722 with a specificity of 82%. A combined biomarker panel of six chemokines CCL2, CCL3, CCL4, CXCL1, CXCL8, and CXCL12 provides a sensitivity of 71% and specificity of 67%. Conclusion The present work demonstrates that the levels of five chemokine-proteins are indicative of underlying disease. We demonstrate technical feasibility and promising clinical performance of a chemokine-based biomarker panel, equivalent to that of other triage options. Further assessment in longitudinal series is now warranted. Methods A panel of 31 chemokines were investigated for expression in routinely taken archived and prospective cervical liquid based cytology (LBC) samples using Human Chemokine Proteomic Array kit. Nine chemokines were further validated using Procartaplex assay on the Luminex platform

Development of an in-house ELISA to detect anti-HPV16-L1 antibodies in serum and dried blood spots

Measuring anti-HPV antibody levels is important for surveillance of the immunological response to both natural infection and vaccination. Here, an ELISA test for measurement of HPV-16L1 antibodies was developed and validated in sera and dried blood spots. An in-house ELISA was developed for measuring anti-HPV-16L1 IgA and IgG levels. The assay was standardized against WHO international standard serum and validated on serum, dried blood spots and cervical liquid based cytology samples from women attending colposcopy clinics in Scotland. Antibody avidity index was also measured in serum samples. The average HPV 16-L1 specific IgG and IgA levels measured in sera, in women attending a routine colposcopy service were 7.3 units/ml and 8.1 units/ml respectively. Significant correlations between serum and dried blood spot eluates for both IgG and IgA were observed indicating that the latter serve as a credible proxy for antibody levels. Average IgG Avidity Index was 35% (95% CI 25%-45%) suggesting previous, historical challenge with natural infection. This ELISA has potential for use in epidemiological and field studies of antibody prevalence and if coupled with avidity measurement may be of use in individual case monitoring of vaccine responses and failures

Changes in HPV prevalence following a national bivalent HPV vaccination programme in Scotland: a 7-year cross-sectional study

Background: On Sept 1, 2008, Scotland launched routine vaccination for human papillomavirus (HPV) types 16 and 18, targeted at 12–13-year-old girls, of whom 92·4% were fully vaccinated in 2008–09. In this study, we report on vaccine effectiveness of the bivalent vaccine in these vaccinated women who attended for routine cervical screening at age 20–21 years. Methods: In this 7-year cross-sectional study (covering birth cohorts 1988–1995), we sampled approximately 1000 samples per year from those attending cervical screening at age 20–21 years and tested each for HPV. By linkage to vaccination records we ascertained prevalence by birth cohort and vaccination status. Estimates of vaccine effectiveness for HPV types 16 and 18, HPV types 31, 33, and 45, other high-risk types, and any HPV were calculated using logistic regression. Findings: In total, 8584 samples were HPV genotyped. Prevalence of HPV types 16 and 18 reduced substantially from 30·0% (95% CI 26·9–33·1) in the 1988 cohort to 4·5% (3·5–5·7) in the 1995 cohort, giving a vaccine effectiveness of 89·1% (85·1–92·3) for those vaccinated at age 12–13 years. All cross-protective types showed significant vaccine effectiveness (HPV type 31, 93·8% [95% CI 83·8–98·5]; HPV type 33, 79·1% [64·2–89·0]; HPV type 45, 82·6% [61·5–93·9]). Unvaccinated individuals born in 1995 had a reduced odds of HPV types 16 and 18 infection compared with those born in 1988 (adjusted odds ratio 0·13 [95% CI 0·06–0·28]) and reduced odds of HPV types 31, 33, and 45 (odds ratio 0·45 [0·23–0·89]). Interpretation: Bivalent vaccination has led to a startling reduction in vaccine and cross-protective HPV types 7 years after vaccination. There is also evidence of herd protection against the vaccine-specific and cross-protective types in unvaccinated individuals born in 1995. These findings should be considered in cost-effectiveness models informing vaccine choice and models to shape the future of cervical screening programmes

Macular pigment and percentage of body fat

PURPOSE. To investigate the relationship between percentage of body fat and macular pigment (MP) optical density. METHODS. One hundred healthy subjects of ages between 22 and 60 years volunteered to participate in this study. MP optical density was measured psychophysically, serum lutein and zeaxanthin were quantified by HPLC, and dietary intake of lutein and zeaxanthin was assessed using a validated food frequency questionnaire. Body fat was measured by dual energy x-ray absorptiometry (DEXA); body mass index (BMI) was also calculated for each subject. Clinical and personal details were recorded, with particular attention directed toward putative risk factors for AMD. RESULTS. There was a significant inverse relationship between the percentage of body fat and MP optical density in males (r ϭ Ϫ0.392, P Ͻ 0.01), and after correcting for age and dietary lutein and zeaxanthin, this inverse relationship remained significant (r ϭ Ϫ0.290, P Ͻ 0.05). The relationship between MP optical density and percentage of body fat in females was inverse, but not significant (r ϭ Ϫ0.197, P ϭ 0.149). A significant and inverse relationship between serum zeaxanthin and percentage of body fat was observed for females only (r ϭ Ϫ0.354, P Ͻ 0.01). Dietary intake of fat was inversely related to serum lutein and zeaxanthin, and significantly so for lutein (r ϭ Ϫ0.256, P Ͻ 0.05). However, dietary fat was unrelated to MP optical density (r ϭ 0.041, P ϭ 0.688). CONCLUSIONS. A relative lack of MP is associated with adiposity in men, and may underlie the association between body fat and risk for AMD progression in males. Further, the processes governing accumulation and/or stabilization of lutein and zeaxanthin in fat tissue appear to differ for males and females. (Invest Ophthalmol Vis Sci. 2004;45:3940 -3950) DOI: 10.1167/iovs.04-0273 A ge-related macular degeneration (AMD), which damages central vision, is the late stage of age-related maculopathy (ARM), and is the leading cause of blindness in elderly people in the Western World. 1 Under physiological conditions, lutein and zeaxanthin accumulate at the macula to the exclusion of all other carotenoids, and are collectively known as macular pigment (MP). The function of MP remains uncertain, but it is believed to reduce chromatic aberration as a result of its absorption spectrum which peaks at 460 nm (blue light). 2 These absorptive characteristics, alone or in combination with the capacity of the retinal carotenoids to quench reactive oxygen intermediates (ROIs), allow the MP to protect the retina from oxidative damage. Although the pathogenesis of AMD remains unclear, there is a growing body of evidence implicating oxidative stress, which refers to tissue damage arising from interaction between the constituent molecules of a tissue and ROIs. The retina is an ideal site for the generation of ROIs because of its high oxygen consumption, its wealth of chromophores, and its exposure to short wavelength visible light. Body fat is of particular interest, because adipose tissue is a major storage organ for carotenoids. 8 Of note, several large population-based studies have found an association between incidence and/or prevalence of AMD and BMI. 9 -13 BMI lends itself as a measure of obesity in epidemiologic studies, largely because it is quick and easy to record. However, the accuracy of BMI (a simple weight to height ratio) as a measure of fat mass, and consequently as a tool for investigating its relationship with carotenoid levels in the blood and retina, remains controversial. -17 The present study was designed to investigate the relationship between MP optical density and percentage of body fat as measured by dual energy x-ray absorptiometry (DEXA), which is the most available direct assessment of fat mass. The relationship between adiposity and serum concentrations of lutein and zeaxanthin was also evaluated. METHODS Subjects One hundred healthy subjects volunteered to participate in this study, which was approved by the Research Ethics Committee of Waterford Regional Hospital and the Ethics Committee of the Waterford Institute of Technology. Informed consent was obtained from each volunteer, and the experimental procedures adhered to the tenets of the Declaration of Helsinki. Subjects were recruited to this single-visit study at the Waterford Institute of Technology by a self-selected sample population who volunteered as a result of posters, newsletters, and word of mouth in the local community. Subjects had to be white between the ages of 20 and 60 years. Volunteers with ocular pathology were excluded. The following details were recorded for each volunteer: demographic data; general health status, with particular attention directed From th

The relationship between cannabis outcome expectancies and cannabis refusal self-efficacy in a treatment population

Background and aims: Self-efficacy beliefs and outcome expectancies are central to Social Cognitive Theory (SCT). Alcohol studies demonstrate the theoretical and clinical utility of applying both SCT constructs. This study examined the relationship between refusal self-efficacy and outcome expectancies in a sample of cannabis users, and tested formal mediational models. Design: Patients referred for cannabis treatment completed a comprehensive clinical assessment, including recently validated cannabis expectancy and refusal self-efficacy scales. Setting: A hospital alcohol and drug out-patient clinic. Participants: Patients referred for a cannabis treatment [n=1115, mean age 26.29, standard deviation (SD) 9.39]. Measurements: The Cannabis Expectancy Questionnaire (CEQ) and Cannabis Refusal Self-Efficacy Questionnaire (CRSEQ) were completed, along with measures of cannabis severity [Severity of Dependence Scale (SDS)] and cannabis consumption. Findings: Positive (β=-0.29,

Risk stratification of cervical disease using detection of human papillomavirus (HPV) E4 protein and cellular MCM protein in clinical liquid based cytology samples

Background: While human papillomavirus (HPV) DNA testing offers high sensitivity for the detection of significant cervical disease, its specificity is suboptimal given the high prevalence of transient HPV infections (CIN1 or less). Biomarkers to identify those suffering from low grade disease from those with high grade disease could save healthcare costs and reduce patient anxiety. Objective: The objective of the present work was to develop and test an immunohistochemistry (IHC)-based dual viral and cellular biomarker strategy which was applicable to liquid based cytology (LBC) samples. Study design: We developed a novel IHC assay for detection of HPV E4 and cellular minichromosome maintenance (MCM) proteins in routinely taken cervical LBC samples using cytospin-prepared slides. The assay was applied to a prospective cohort of Scottish women referred to a colposcopy clinic due to preceding cytological abnormalities. The performance of the biomarkers for detection of clinically insignificant (CIN1 or less) versus significant disease was determined. Results: A total of 81 women were recruited representing 64 cases of <=CIN1 and 28 of CIN2 + . Biomarker performance relative to histopathology outcomes showed high levels of MCM detection was significantly associated with CIN2+ (p = 0.03) while E4 was detected more frequently in <=CIN1 (p = 0.06). Conclusions: Combined detection of a host proliferation marker and a marker of viral gene expression could allow triage of cases of clinically insignificant disease prior to colposcopy. However, there was overlap between distributions of MCM levels in CIN2+ and <=CIN1 suggesting that additional biomarkers would be required for improved specificity. Combined with cytospin-prepared slides this approach could provide a means of risk stratification of disease in low resource settings

Human Papilloma Virus (HPV) Oral Prevalence in Scotland (HOPSCOTCH):a feasibility study in dental settings

The purpose of this study was to test the feasibility of undertaking a full population investigation into the prevalence, incidence, and persistence of oral Human Papilloma Virus (HPV) in Scotland via dental settings. Male and female patients aged 16-69 years were recruited by Research Nurses in 3 primary care and dental outreach teaching centres and 2 General Dental Practices (GDPs), and by Dental Care Teams in 2 further GDPs. Participants completed a questionnaire (via an online tablet computer or paper) with socioeconomic, lifestyle, and sexual history items; and were followed up at 6-months for further questionnaire through appointment or post/online. Saline oral gargle/rinse samples, collected at baseline and follow-up, were subject to molecular HPV genotyping centrally. 1213 dental patients were approached and 402 individuals consented (participation rate 33.1%). 390 completed the baseline questionnaire and 380 provided a baseline oral specimen. Follow-up rate was 61.6% at 6 months. While recruitment was no different in Research Nurse vs Dental Care Team models the Nurse model ensured more rapid recruitment. There were relatively few missing responses in the questionnaire and high levels of disclosure of risk behaviours (99% answered some of the sexual history questions). Data linkage of participant data to routine health records including HPV vaccination data was successful with 99.1% matching. Oral rinse/gargle sample collection and subsequent HPV testing was feasible. Preliminary analyses found over 95% of samples to be valid for molecular HPV detection prevalence of oral HPV infection of 5.5% (95%CI 3.7, 8.3). It is feasible to recruit and follow-up dental patients largely representative / reflective of the wider population, suggesting it would be possible to undertake a study to investigate the prevalence, incidence, and determinants of oral HPV infection in dental settings