Functional and genetic analysis of haplotypic sequence variation at the nicastrin genomic locus

Nicastrin (NCSTN) is a component of the ?-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn(-/-)) cells and clonal NCSTN-BAC(+)/Ncstn(-/-) cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued ?-secretase activity and amyloid beta (Aß) production in NCSTN-BAC(+)/Ncstn(-/-) lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease.

Obesity associated severe asthma represents a distinct clinical phenotype - Analysis of the British Thoracic Society Difficult Asthma Registry patient cohort according to body mass index

BACKGROUND: Obesity has emerged as a risk factor for the development of asthma and it may also influence asthma control and airways inflammation. However, the role of obesity in severe asthma remains unclear. OBJECTIVE: To explore the association between obesity (defined by BMI) and severe asthma. METHODS: Data from the National Registry for dedicated UK Difficult Asthma Services were used to compare patient demographics, disease characteristics and healthcare utilisation between three body mass index (BMI) categories (normal weight: 18.5 -24.99, overweight: 25 -29.99, obese: =30) in a well characterised group of severe asthmatic adults. RESULTS: The study population consisted of 666 severe asthmatics with a median BMI of 29.8 (interquartile range 22.5 -34.0). The obese group exhibited greater asthma medication requirements in terms of maintenance corticosteroid therapy (48.9% versus 40.4% and 34.5% in the overweight and normal weight groups, respectively), steroid burst therapy and short-acting ß2-agonist (SABA) use per day. Significant differences were seen with gastro-oesophageal reflux disease (GORD) (53.9% versus 48.1% and 39.7% in the overweight and normal weight groups, respectively) and proton pump inhibitor (PPI) use. Bone density scores were higher in the obese group, whilst pulmonary function testing revealed a reduced FVC and raised Kco. Serum IgE levels decreased with increasing BMI and the obese group were more likely to report eczema, but less likely to have a history of nasal polyps. CONCLUSIONS: Severe asthmatics display particular characteristics according to BMI that support the view that obesity associated severe asthma may represent a distinct clinical phenotype.1Royal Brompton Hospital, London, UK;2Department of Computing, Imperial College, UK3Airways Disease, National Heart & Lung Institute, Imperial College, UK;4Centre for infection and immunity, Queen's University of Belfast, UK;5University of Leicester, UK;6The University of Manchester and University Hospital of South Manchester, UK;7Birmingham Heartlands Hospital, University of Birmingham, UK;8Gartnavel General Hospital, University of Glasgow, UK;9Glasgow Royal Infirmary, Glasgow, UKCorrespondence: Dr Andrew N. Menzies-Gow, Royal Brompton Hospital, Fulham Road, London SW3 6HP.

Score for neonatal acute physiology-II and neonatal pain predict corticospinal tract development in premature newborns

Premature infants are at risk for adverse motor outcomes, including cerebral palsy and developmental coordination disorder. The purpose of this study was to examine the relationship of antenatal, perinatal, and postnatal risk factors for abnormal development of the corticospinal tract, the major voluntary motor pathway, during the neonatal period. In a prospective cohort study, 126 premature neonates (24-32 weeks' gestational age) underwent serial brain imaging near birth and at term-equivalent age. With diffusion tensor tractography, mean diffusivity and fractional anisotropy of the corticospinal tract were measured to reflect microstructural development. Generalized estimating equation models examined associations of risk factors on corticospinal tract development. The perinatal risk factor of greater early illness severity (as measured by the Score for Neonatal Acute Physiology-II [SNAP-II]) was associated with a slower rise in fractional anisotropy of the corticospinal tract (P = 0.02), even after correcting for gestational age at birth and postnatal risk factors (P = 0.009). Consistent with previous findings, neonatal pain adjusted for morphine and postnatal infection were also associated with a slower rise in fractional anisotropy of the corticospinal tract (P = 0.03 and 0.02, respectively). Lessening illness severity in the first hours of life might offer potential to improve motor pathway development in premature newborns.

Maternal Religiosity, Family Resources and Stressors, and Parent-Child Attachment Security in Northern Ireland

This study explores the associations between mothers' religiosity, and families' and children's functioning in a stratified random sample of 695 Catholic and Protestant motherchild dyads in socially deprived areas in Belfast, Northern Ireland, a region which has experienced centuries of sectarian conflict between Protestant Unionists and Catholics Nationalists. Findings based on mother and child surveys indicated that even in this context of historical political violence associated with religious affiliation, mothers' religiosity played a consistently positive role, including associations with multiple indicators of better family functioning (i.e., more cohesion and behavioral control and less conflict, psychological distress, and adjustment problems) and greater parentchild attachment security. Mothers' religiosity also moderated the association between parentchild attachment security and family resources and family stressors, enhancing positive effects of cohesion and mother behavioral control on motherchild attachment security, and providing protection against risks associated with mothers' psychological distress. Findings are discussed in terms of implications for understanding the role of religiosity in serving as a protective or risk factor for children and families.

Lipopolysaccharide-binding protein and CD14 are increased in the bronchoalveolar lavage fluid of smokers

Lipopolysaccharide-binding protein (LBP) and CD14 contribute to the recognition of pathogens by cells, which triggers the activation of defence responses. Smoking is a risk factor for the development of chronic obstructive pulmonary disease (COPD) and respiratory infections. The current authors theorised that levels of LBP and CD14 in the lungs of smokers would be higher than those in the lungs of never-smokers. These elevated levels could affect host responses upon infection. LBP, soluble CD14 (sCD14) and interleukin (IL)-8 were detected by ELISA. Nuclear factor (NF)- ?B, p38 and the inhibitor I?Ba were studied by immunoassays. Gene expression was assessed by RT-PCR. Bronchoalveolar lavage levels of LBP and CD14 were significantly higher in smokers and COPD patients than in never-smokers, whereas levels of both proteins were not significantly different between smokers and COPD patients. IL-6, IL-1ß5 and cigarette smoke condensate induced the expression of LBP and CD14 by airway epithelial cells. LBP and sCD14 inhibited the nontypeable Haemophilus influenzae (NTHi)-dependent secretion of IL-8 and the activation of NF-?B and p38 mitogen-activated protein kinase signalling pathways but they increased the internalisation of NTHi by airway epithelial cells. Thus, in the inflamed airways of smokers both proteins could contribute to inhibit bacteria-dependent cellular activation without compromising the internalisation of pathogens by airway cells. Copyright©ERS Journals Ltd 2009.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

Identifying research priorities on infections in older adults:Proceedings of an interdisciplinary workshop

Background: Infections pose a substantial burden to the health of older adults. In this report, we describe the proceedings of a workshop to formulate and prioritize research questions about infections in older adults using an interdisciplinary approach. Methods: Researchers from four sectors (basic science, clinical sciences, health services and epidemiology/determinants of health) and representatives from various Canadian local, provincial, and federal stakeholder groups were invited to a two-day workshop. Five multi-disciplinary groups and stakeholders from each of three healthcare settings (long term, acute care and community) discussed research priorities for each of the settings. Five to ten research questions were identified for each setting. Results: The research questions proposed ranged from risk factors and outcomes for different infections to the effect of nutrition on infection and the role of alternative and complementary medicine in treating infections. Health service issues included barriers to immunization, prolongation of hospital length of stay by infection, use of care paths for managing infections, and decision-making in determining the site of care for individuals with infections. Clinical questions included risk factor assessment for infection, the effectiveness of preventative strategies, and technology evaluation. Epidemiologic issues included the challenge of achieving a better understanding of respiratory infections in the community and determining the prevalence of colonization with multi-resistant bacteria. Conclusions: The questions are of direct relevance to researchers in a wide variety of fields. Bringing together a multi-disciplinary group of researchers to frame and prioritize research questions about aging is feasible, participants valued the opinions of people working in other areas.

Environmental tobacco smoke exposure and eye disease

Aim: To undertake a systematic review of the literature on the effect of environmental tobacco smoke (ETS) and eye disease. Methods: Medline (1950-January Week 2 2007), EMBASE (1980 to 2007 Week 07), SCOPUS and Science Direct were searched on ETS exposure and eye disease using various combinations of the following terms: passive smoking, environmental tobacco smoke, sidestream smoke, involuntary smoking, secondhand smoke; with eye, conjunctiva, sclera, episclera, cornea, lens, iris, retina, choroid, uvea, optic nerve, uveitis, iritis, blindness, visual loss, cataract, thyroid eye disease, conjunctivitis, age-related macular degeneration, dry eye, tears. The above terms were also used to search abstracts published on The Association for Research in Vision and Ophthalmology Annual Meeting abstracts, from 1995 to 2006, and the grey literature, including PhD and MSc theses/dissertations. A search was further conducted specifically on eye diseases where active smoking has been proposed to be a risk factor, including age-related macular degeneration, Graves ophthalmology, glaucoma, uveitis, refractive errors, strabismus, tobacco-alcohol amblyopia, non-arteritic ischaemic optic neuropathy, Leber optic neuropathy and diabetic retinopathy. Given the scarce number of studies found through the above search, all articles found on ETS and eye disease were included in this review. Results: Seven studies evaluated the possible relationship between ETS and an eye disease. These studies referred to refractive errors in children (n = 2), cataract (n = 1), age-related macular degeneration (n = 3) and Grave ophthalmopathy (n = 1). The data available were insufficient to establish conclusive relationships between ETS and these eye diseases. Conclusion: Very scarce data exist in the literature on the effect of ETS on diseases of the eye. It seems appropriate that ETS should be included in future studies addressing the effect of smoking on eye disease.

Association Analysis of Dyslipidemia-Related Genes in Diabetic Nephropathy

Type 1 diabetes (T1D) increases risk of the development of microvascular complications and cardiovascular disease (CVD). Dyslipidemia is a common risk factor in the pathogenesis of both CVD and diabetic nephropathy (DN), with CVD identified as the primary cause of death in patients with DN. In light of this commonality, we assessed single nucleotide polymorphisms (SNPs) in thirty-seven key genetic loci previously associated with dyslipidemia in a T1D cohort using a casecontrol design. SNPs (n = 53) were genotyped using Sequenom in 1467 individuals with T1D (718 cases with proteinuric nephropathy and 749 controls without nephropathy i.e. normal albumin excretion). Cases and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK to compare allele frequencies in cases and controls. In a sensitivity analysis, samples from control individuals with reduced renal function (estimated glomerular filtration rate,60 ml/min/1.73 m2) were excluded. Correction for multiple testing was performed by permutation testing. A total of 1394 samples passed quality control filters. Following regression analysis adjusted by collection center, gender, duration of diabetes, and average HbA1c, two SNPs were significantly associated with DN. rs4420638 in the APOC1 region (odds ratio [OR] = 1.51; confidence intervals [CI]: 1.19–1.91; P = 0.001) and rs1532624 in CETP (OR = 0.82; CI: 0.69–0.99; P = 0.034); rs4420638 was also significantly associated in a sensitivity analysis (P = 0.016) together with rs7679 (P = 0.027). However, no association was significant following correction for multiple testing. Subgroup analysis of end-stage renal disease status failed to reveal any association. Our results suggest common variants associated with dyslipidemia are not strongly associated with DN in T1D among white individuals. Our findings, cannot entirely exclude these key genes which are central to the process of dyslipidemia, from involvement in DN pathogenesis as our study had limited power to detect variants of small effect size. Analysis in larger independent cohorts is required.

The United Kingdom Glaucoma Treatment Study:a multicenter, randomized, placebo-controlled clinical trial: design and methodology

Elevated intraocular pressure (IOP) is a major risk factor for the deterioration of open-angle glaucoma (OAG); medical IOP reduction is the standard treatment, yet no randomized placebo-controlled study of medical IOP reduction has been undertaken previously. The United Kingdom Glaucoma Treatment Study (UKGTS) tests the hypothesis that treatment with a topical prostaglandin analog, compared with placebo, reduces the frequency of visual field (VF) deterioration events in OAG patients by 50% over a 2-year period.

Biomarkers in diabetes:hemoglobin A1c, vascular and tissue markers

Biomarkers are conventionally defined as "biological molecules that represent health and disease states." They typically are measured in readily available body fluids (blood or urine), lie outside the causal pathway, are able to detect subclinical disease, and are used to monitor clinical and subclinical disease burden and response to treatments. Biomarkers can be "direct" endpoints of the disease itself, or "indirect" or surrogate endpoints. New technologies (such as metabolomics, proteomics, genomics) bring a wealth of opportunity to develop new biomarkers. Other new technologies enable the development of nonmolecular, functional, or biophysical tissue-based biomarkers. Diabetes mellitus is a complex disease affecting almost every tissue and organ system, with metabolic ramifications extending far beyond impaired glucose metabolism. Biomarkers may reflect the presence and severity of hyperglycemia (ie, diabetes itself) or the presence and severity of the vascular complications of diabetes. Illustrative examples are considered in this brief review. In blood, hemoglobin A1c (HbA1c) may be considered as a biomarker for the presence and severity of hyperglycemia, implying diabetes or prediabetes, or, over time, as a "biomarker for a risk factor," ie, hyperglycemia as a risk factor for diabetic retinopathy, nephropathy, and other vascular complications of diabetes. In tissues, glycation and oxidative stress resulting from hyperglycemia and dyslipidemia lead to widespread modification of biomolecules by advanced glycation end products (AGEs). Some of these altered species may serve as biomarkers, whereas others may lie in the causal pathway for vascular damage. New noninvasive technologies can detect tissue damage mediated by AGE formation: these include indirect measures such as pulse wave analysis (a marker of vascular dysfunction) and more direct markers such as skin autofluorescence (a marker of long-term accumulation of AGEs). In the future, we can be optimistic that new blood and tissue-based biomarkers will enable the detection, prevention, and treatment of diabetes and its complications long before overt disease develops.

Treatment approaches for diabetes and dyslipidemia

Dyslipidemia is an important risk factor for cardiovascular complications in persons with diabetes. Low-density lipoprotein-cholesterol (LDL-C) is the 'cornerstone' for assessment of lipoprotein-associated risk. However, LDL-C levels do not reflect the classic 'diabetic dyslipidemia' of hypertriglyceridemia and low high-density lipoprotein-cholesterol (HDL-C). Measurements of plasma apolipoprotein B100 concentrations and non-HDL-C may improve the definition of dyslipidemia. Statins, nicotinic acid and fibrates have roles in treating dyslipidemia in diabetes. Residual risk (i.e. risk that persists after correction of 'conventional' plasma lipoprotein abnormalities) is a new concept in the role of dyslipidemia in the pathogenesis of diabetic vascular complications. For example, regardless of plasma levels, lipoprotein extravasation through a leaking retinal blood barrier and subsequent modification may be crucial in the development of diabetic retinopathy. The current approach to the management of dyslipidemia in diabetes is briefly summarized, followed by a discussion of new concepts of residual risk and emerging lipoprotein-related mechanisms for vascular disease in diabetes.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)