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1.  Persistent and Compartmentalised Disruption of Dendritic Cell Subpopulations in the Lung following Influenza A Virus Infection 
PLoS ONE  2014;9(11):e111520.
Immunological homeostasis in the respiratory tract is thought to require balanced interactions between networks of dendritic cell (DC) subsets in lung microenvironments in order to regulate tolerance or immunity to inhaled antigens and pathogens. Influenza A virus (IAV) poses a serious threat of long-term disruption to this balance through its potent pro-inflammatory activities. In this study, we have used a BALB/c mouse model of A/PR8/34 H1N1 Influenza Type A Virus infection to examine the effects of IAV on respiratory tissue DC subsets during the recovery phase following clearance of the virus. In adult mice, we found differences in the kinetics and activation states of DC residing in the airway mucosa (AMDC) compared to those in the parenchymal lung (PLDC) compartments. A significant depletion in the percentage of AMDC was observed at day 4 post-infection that was associated with a change in steady-state CD11b+ and CD11b− AMDC subset frequencies and significantly elevated CD40 and CD80 expression and that returned to baseline by day 14 post-infection. In contrast, percentages and total numbers of PLDC were significantly elevated at day 14 and remained so until day 21 post-infection. Accompanying this was a change in CD11b+and CD11b− PLDC subset frequencies and significant increase in CD40 and CD80 expression at these time points. Furthermore, mice infected with IAV at 4 weeks of age showed a significant increase in total numbers of PLDC, and increased CD40 expression on both AMDC and PLDC, when analysed as adults 35 days later. These data suggest that the rate of recovery of DC populations following IAV infection differs in the mucosal and parenchymal compartments of the lung and that DC populations can remain disrupted and activated for a prolonged period following viral clearance, into adulthood if infection occurred early in life.
PMCID: PMC4232261  PMID: 25398128
2.  Polymorphism and resolution of oncogene promoter quadruplex-forming sequences 
Organic & biomolecular chemistry  2011;9(22):7633-7637.
We report the separation of several quadruplex species formed by ten promoter sequences by Size Exclusion Chromatography (SEC). Modification at the 5’ or 3’ ends or in loop regions of quadruplex forming sequences has become the standard technique for dealing with quadruplex polymorphism. However, conformations produced employing this method or by other means of artificially shifting the equilibrium may not represent the species that are present in vivo. This method enables an unperturbed view of the structural polymorphism inherent to quadruplex formation. Separation via SEC facilitates studies on quadruplex structure and biophysical properties without the need for sequence modification.
PMCID: PMC3962748  PMID: 21938285
4.  Elucidation of Pathways Driving Asthma Pathogenesis: Development of a Systems-Level Analytic Strategy 
Asthma is a genetically complex, chronic lung disease defined clinically as episodic airflow limitation and breathlessness that is at least partially reversible, either spontaneously or in response to therapy. Whereas asthma was rare in the late 1800s and early 1900s, the marked increase in its incidence and prevalence since the 1960s points to substantial gene × environment interactions occurring over a period of years, but these interactions are very poorly understood (1–6). It is widely believed that the majority of asthma begins during childhood and manifests first as intermittent wheeze. However, wheeze is also very common in infancy and only a subset of wheezy children progress to persistent asthma for reasons that are largely obscure. Here, we review the current literature regarding causal pathways leading to early asthma development and chronicity. Given the complex interactions of many risk factors over time eventually leading to apparently multiple asthma phenotypes, we suggest that deeply phenotyped cohort studies combined with sophisticated network models will be required to derive the next generation of biological and clinical insights in asthma pathogenesis.
PMCID: PMC4172064  PMID: 25295037
allergy; asthma; systems biology; virus infection; birth cohort; childhood; immune function; epidemiology
5.  Not All G-Quadruplexes are Created Equally: An Investigation of the Structural Polymorphism of the c-Myc G-Quadruplex-Forming Sequence and its Interaction with the Porphyrin meso-Tetra(N-methyl-4-pyridyl)porphine 
Organic & biomolecular chemistry  2012;10(47):9393-9404.
G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III1 promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail.
PMCID: PMC3501587  PMID: 23108607
6.  Safety and Immunogenicity of Neonatal Pneumococcal Conjugate Vaccination in Papua New Guinean Children: A Randomised Controlled Trial 
PLoS ONE  2013;8(2):e56698.
Approximately 826,000 children, mostly young infants, die annually from invasive pneumococcal disease. A 6-10-14-week schedule of pneumococcal conjugate vaccine (PCV) is efficacious but neonatal PCV may provide earlier protection and better coverage. We conducted an open randomized controlled trial in Papua New Guinea to compare safety, immunogenicity and priming for memory of 7-valent PCV (PCV7) given in a 0-1-2-month (neonatal) schedule with that of the routine 1-2-3-month (infant) schedule.
We randomized 318 infants at birth to receive PCV7 in the neonatal or infant schedule or no PCV7. All infants received 23-valent pneumococcal polysaccharide vaccine (PPV) at age 9 months. Serotype-specific serum IgG for PCV7 (VT) serotypes and non-VT serotypes 2, 5 and 7F were measured at birth and 2, 3, 4, 9, 10 and 18 months of age. Primary outcomes were geometric mean concentrations (GMCs) and proportions with concentration ≥0.35 µg/ml of VT serotype-specific pneumococcal IgG at age 2 months and one month post-PPV.
We enrolled 101, 105 and 106 infants, respectively, into neonatal, infant and control groups. Despite high background levels of maternally derived antibody, both PCV7 groups had higher GMCs than controls at age 2 months for serotypes 4 (p<0.001) and 9V (p<0.05) and at age 3 months for all VTs except 6B. GMCs for serotypes 4, 9V, 18C and 19F were significantly higher (p<0.001) at age 2 months in the neonatal (one month post-dose2 PCV7) than in the infant group (one month post-dose1 PCV7). PPV induced significantly higher VT antibody responses in PCV7-primed than unprimed infants, with neonatal and infant groups equivalent. High VT and non-VT antibody concentrations generally persisted to age 18 months.
PCV7 is well-tolerated and immunogenic in PNG neonates and young infants and induces immunologic memory to PPV booster at age 9 months with antibody levels maintained to age 18 months.
Trial Registration NCT00219401NCT00219401
PMCID: PMC3579820  PMID: 23451070
7.  Gene-Vitamin D Interactions on Food Sensitization: A prospective birth cohort study 
Allergy  2011;66(11):1442-1448.
It has been hypothesized that vitamin D deficiency (VDD) contributes to the development of food sensitization (FS) and then food allergy. However, the epidemiological evidence is conflicting. We aim to examine if cord blood VDD is associated with FS and if such association can be modified by genetic variants in a prospective birth cohort.
This study included 649 children who were enrolled at birth and followed from birth onward at the Boston Medical Center. We defined VDD as cord blood 25(OH)D < 11ng/ml, and FS as specific IgE ≥ 0.35kUA/L to any of eight common food allergens in early childhood. We genotyped potentially functional single nucleotide polymorphisms (SNPs) in 11 genes known to be involved in regulating IgE and 25(OH)D concentrations. Logistic regressions were used to test the effects of VDD on FS individually and jointly with SNPs.
Among the 649 children, 44% had VDD and 37% had FS. When examined alone, VDD was not associated with FS. When examined jointly with SNPs, a significant interaction between IL4 gene polymorphism (rs2243250) and VDD (pinteraction=0.003, pFDR=0.10) was found: VDD increased the risk of FS among children carrying CC/CT genotypes (OR=1.79, 95%CI: 1.15–2.77). Similar but weaker interactions were observed for SNPs in MS4A2 (rs512555), FCER1G (rs2070901), and CYP24A1 (rs2762934). When all four SNPs were simultaneously considered, a strong gene-VDD interaction was evident (pinteraction=9×10−6).
Our data demonstrate that VDD may increase the risk of FS among individuals with certain genotypes, providing evidence of gene-vitamin D interaction on FS.
PMCID: PMC3189275  PMID: 21819409
cord blood plasma 25(OH)D; food sensitization; gene-vitamin D deficiency interaction; SNP
8.  Airway Epithelial Cells Condition Dendritic Cells to Express Multiple Immune Surveillance Genes 
PLoS ONE  2012;7(9):e44941.
Increasing evidence suggests that crosstalk between airway epithelial cells (AEC) and adjacent dendritic cells (DC) tightly regulates airway mucosal DC function in steady state. AEC are known to express multiple immmuno-modulatory factors, though detailed information on how this influences human DC function remains incomplete. We recently demonstrated using an in vitro coculture model that AEC alter differentiation of monocytes into DC in a manner that inhibits expression of potentially damaging Th2 effector function. In the current study, we have extended these findings to examine other aspects of DC function. Using micro-array technology we show that multiple genes important for immune surveillance are significantly over expressed in purified AEC-conditioned DC, compared to control DC. These findings were confirmed by quantitative real time PCR or flow cytometry in an independent sample set. In particular, AEC-conditioned DC showed selective upregulation of chemokines that recruit Th1 cells, but minimal change in chemokines linked to Th2 cell recruitment. AEC-conditioned DC were also characterized by enhanced expression of complement family genes (C1QB, C2, CD59 and SERPING1), Fcγ receptor genes (FCGR1A, FCGR2A, FCGR2B and FCGR2C), signaling lymphocytic activation molecule family member 1 (SLAM), programmed death ligands 1 and 2, CD54 and CD200R1, relative to control DC. These findings suggest that AEC conditioning facilitates the capacity of DC to react to danger signals, to enhance leukocyte recruitment, especially of Th1 effector cells, and to interact with other immune cell populations while minimizing the risks of excessive inflammation leading to tissue damage.
PMCID: PMC3439377  PMID: 22984588
Paternoster, Lavinia | Standl, Marie | Chen, Chih-Mei | Ramasamy, Adaikalavan | Bønnelykke, Klaus | Duijts, Liesbeth | Ferreira, Manuel A | Alves, Alexessander Couto | Thyssen, Jacob P | Albrecht, Eva | Baurecht, Hansjörg | Feenstra, Bjarke | Sleiman, Patrick MA | Hysi, Pirro | Warrington, Nicole M | Curjuric, Ivan | Myhre, Ronny | Curtin, John A | Groen-Blokhuis, Maria M | Kerkhof, Marjan | Sääf, Annika | Franke, Andre | Ellinghaus, David | Fölster-Holst, Regina | Dermitzakis, Emmanouil | Montgomery, Stephen B | Prokisch, Holger | Heim, Katharina | Hartikainen, Anna-Liisa | Pouta, Anneli | Pekkanen, Juha | Blakemore, Alexandra IF | Buxton, Jessica L | Kaakinen, Marika | Duffy, David L | Madden, Pamela A | Heath, Andrew C | Montgomery, Grant W | Thompson, Philip J | Matheson, Melanie C | Le Souëf, Peter | Pourcain, Beate St | Smith, George Davey | Henderson, John | Kemp, John P | Timpson, Nicholas J | Deloukas, Panos | Ring, Susan M | Wichmann, H-Erich | Müller-Nurasyid, Martina | Novak, Natalija | Klopp, Norman | Rodríguez, Elke | McArdle, Wendy | Linneberg, Allan | Menné, Torkil | Nohr, Ellen A | Hofman, Albert | Uitterlinden, André G | van Duijn, Cornélia M | Rivadeneira, Fernando | de Jongste, Johan C | van der Valk, Ralf JP | Wjst, Matthias | Jogi, Rain | Geller, Frank | Boyd, Heather A | Murray, Jeffrey C | Kim, Cecilia | Mentch, Frank | March, Michael | Mangino, Massimo | Spector, Tim D | Bataille, Veronique | Pennell, Craig E | Holt, Patrick G | Sly, Peter | Tiesler, Carla MT | Thiering, Elisabeth | Illig, Thomas | Imboden, Medea | Nystad, Wenche | Simpson, Angela | Hottenga, Jouke-Jan | Postma, Dirkje | Koppelman, Gerard H | Smit, Henriette A | Söderhäll, Cilla | Chawes, Bo | Kreiner-Møller, Eskil | Bisgaard, Hans | Melén, Erik | Boomsma, Dorret I | Custovic, Adnan | Jacobsson, Bo | Probst-Hensch, Nicole M | Palmer, Lyle J | Glass, Daniel | Hakonarson, Hakon | Melbye, Mads | Jarvis, Deborah L | Jaddoe, Vincent WV | Gieger, Christian | Strachan, David P | Martin, Nicholas G | Jarvelin, Marjo-Riitta | Heinrich, Joachim | Evans, David M | Weidinger, Stephan
Nature genetics  2011;44(2):187-192.
Atopic dermatitis (AD) is a common chronic skin disease with high heritability. Apart from filaggrin (FLG), the genes influencing AD are largely unknown. We conducted a genome-wide association meta-analysis of 5,606 cases and 20,565 controls from 16 population-based cohorts and followed up the ten most strongly associated novel markers in a further 5,419 cases and 19,833 controls from 14 studies. Three SNPs met genome-wide significance in the discovery and replication cohorts combined: rs479844 upstream of OVOL1 (OR=0.88, p=1.1×10−13) and rs2164983 near ACTL9 (OR=1.16, p=7.1×10−9), genes which have been implicated in epidermal proliferation and differentiation, as well as rs2897442 in KIF3A within the cytokine cluster on 5q31.1 (OR=1.11, p=3.8×10−8). We also replicated the FLG locus and two recently identified association signals at 11q13.5 (rs7927894, p=0.008) and 20q13.3 (rs6010620, p=0.002). Our results underline the importance of both epidermal barrier function and immune dysregulation in AD pathogenesis.
PMCID: PMC3272375  PMID: 22197932
10.  Gene Polymorphisms, Breastfeeding and Development of Food Sensitization in Early Childhood 
The impact of breastfeeding on the development of allergic disease is uncertain. There are no data that show whether this relationship varies by individual genotypes.
To evaluate the effect of breastfeeding and gene-breastfeeding interactions on food sensitization (FS) in a prospective U.S. birth cohort.
This study included 970 children who were prospectively followed since birth. Breastfeeding history was obtained from a standardized questionnaire interview. FS was defined as specific IgE ≥0.35 kUA/L to any of eight common food allergens. Eighty-eight potentially functional SNPs were genotyped from 18 genes involved in innate immunity or TH1/TH2 balance. Logistic regression models were used to test the effects of breastfeeding and gene-breastfeeding interactions on FS, with adjustment for pertinent covariates.
Children who were ever breastfed (n=739), including exclusively breastfed children, were at a 1.5 (95%CI=1.1-2.1, p=0.019) times higher risk of FS than never breastfed children (n=231). This association was significantly modified by rs425648 in the IL12RB1gene (pinteraction=0.0007): breastfeeding increased the risk of FS (OR=2.0, 95%CI=1.4-3.1, p= 0.0005) in children carrying the GG genotype but decreased the risk (OR=0.6, 95%CI=0.3-1.4, p=0.252) in children carrying the GT/TT genotype. Similar interactions were observed for SNPs in the TLR9 (rs352140) and TSLP (rs3806933) genes. The interaction between the combined genotypes of the three SNPs and breastfeeding on FS was even stronger (pinteraction<10-5).
Our data suggest that the effect of breastfeeding on FS was modified by SNPs in the IL12RB1, TLR9, and TSLP genes both individually and jointly. Our findings underscore the importance of considering individual genetic variations in assessing this relationship.
PMCID: PMC3149737  PMID: 21689850
Breastfeeding; food sensitization; gene-environment interaction
11.  Ontogeny of Toll-Like and NOD-Like Receptor-Mediated Innate Immune Responses in Papua New Guinean Infants 
PLoS ONE  2012;7(5):e36793.
Studies addressing the ontogeny of the innate immune system in early life have reported mainly on Toll-like receptor (TLR) responses in infants living in high-income countries, with little or even no information on other pattern recognition receptors or on early life innate immune responses in children living under very different environmental conditions in less-developed parts of the world. In this study, we describe whole blood innate immune responses to both Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor agonists including the widely used vaccine adjuvant ‘alum’ in a group of Papua New Guinean infants aged 1–3 (n = 18), 4–6 (n = 18), 7–12 (n = 21) and 13–18 (n = 10) months old. Depending on the ligands and cytokines studied, different age-related patterns were found: alum-induced IL-1β and CXCL8 responses were found to significantly decline with increasing age; inflammatory (IL-6, IL-1β, IFN-γ) responses to TLR2 and TLR3 agonists increased; and IL-10 responses remained constant or increased during infancy, while TNF-α responses either declined or remained the same. We report for the first time that whole blood innate immune responses to the vaccine adjuvant alum decrease with age in infancy; a finding that may imply that the adjuvant effect of alum in pediatric vaccines could be age-related. Our findings further suggest that patterns of innate immune development may vary between geographically diverse populations, which in line with the ‘hygiene hypothesis’ particularly involves persistence of innate IL-10 responses in populations experiencing higher infectious pressure.
PMCID: PMC3359332  PMID: 22649499
12.  Genome-wide association and large scale follow-up identifies 16 new loci influencing lung function 
Artigas, María Soler | Loth, Daan W | Wain, Louise V | Gharib, Sina A | Obeidat, Ma’en | Tang, Wenbo | Zhai, Guangju | Zhao, Jing Hua | Smith, Albert Vernon | Huffman, Jennifer E | Albrecht, Eva | Jackson, Catherine M | Evans, David M | Cadby, Gemma | Fornage, Myriam | Manichaikul, Ani | Lopez, Lorna M | Johnson, Toby | Aldrich, Melinda C | Aspelund, Thor | Barroso, Inês | Campbell, Harry | Cassano, Patricia A | Couper, David J | Eiriksdottir, Gudny | Franceschini, Nora | Garcia, Melissa | Gieger, Christian | Gislason, Gauti Kjartan | Grkovic, Ivica | Hammond, Christopher J | Hancock, Dana B | Harris, Tamara B | Ramasamy, Adaikalavan | Heckbert, Susan R | Heliövaara, Markku | Homuth, Georg | Hysi, Pirro G | James, Alan L | Jankovic, Stipan | Joubert, Bonnie R | Karrasch, Stefan | Klopp, Norman | Koch, Beate | Kritchevsky, Stephen B | Launer, Lenore J | Liu, Yongmei | Loehr, Laura R | Lohman, Kurt | Loos, Ruth JF | Lumley, Thomas | Al Balushi, Khalid A | Ang, Wei Q | Barr, R Graham | Beilby, John | Blakey, John D | Boban, Mladen | Boraska, Vesna | Brisman, Jonas | Britton, John R | Brusselle, Guy G | Cooper, Cyrus | Curjuric, Ivan | Dahgam, Santosh | Deary, Ian J | Ebrahim, Shah | Eijgelsheim, Mark | Francks, Clyde | Gaysina, Darya | Granell, Raquel | Gu, Xiangjun | Hankinson, John L | Hardy, Rebecca | Harris, Sarah E | Henderson, John | Henry, Amanda | Hingorani, Aroon D | Hofman, Albert | Holt, Patrick G | Hui, Jennie | Hunter, Michael L | Imboden, Medea | Jameson, Karen A | Kerr, Shona M | Kolcic, Ivana | Kronenberg, Florian | Liu, Jason Z | Marchini, Jonathan | McKeever, Tricia | Morris, Andrew D | Olin, Anna-Carin | Porteous, David J | Postma, Dirkje S | Rich, Stephen S | Ring, Susan M | Rivadeneira, Fernando | Rochat, Thierry | Sayer, Avan Aihie | Sayers, Ian | Sly, Peter D | Smith, George Davey | Sood, Akshay | Starr, John M | Uitterlinden, André G | Vonk, Judith M | Wannamethee, S Goya | Whincup, Peter H | Wijmenga, Cisca | Williams, O Dale | Wong, Andrew | Mangino, Massimo | Marciante, Kristin D | McArdle, Wendy L | Meibohm, Bernd | Morrison, Alanna C | North, Kari E | Omenaas, Ernst | Palmer, Lyle J | Pietiläinen, Kirsi H | Pin, Isabelle | Polašek, Ozren | Pouta, Anneli | Psaty, Bruce M | Hartikainen, Anna-Liisa | Rantanen, Taina | Ripatti, Samuli | Rotter, Jerome I | Rudan, Igor | Rudnicka, Alicja R | Schulz, Holger | Shin, So-Youn | Spector, Tim D | Surakka, Ida | Vitart, Veronique | Völzke, Henry | Wareham, Nicholas J | Warrington, Nicole M | Wichmann, H-Erich | Wild, Sarah H | Wilk, Jemma B | Wjst, Matthias | Wright, Alan F | Zgaga, Lina | Zemunik, Tatijana | Pennell, Craig E | Nyberg, Fredrik | Kuh, Diana | Holloway, John W | Boezen, H Marike | Lawlor, Debbie A | Morris, Richard W | Probst-Hensch, Nicole | Kaprio, Jaakko | Wilson, James F | Hayward, Caroline | Kähönen, Mika | Heinrich, Joachim | Musk, Arthur W | Jarvis, Deborah L | Gläser, Sven | Järvelin, Marjo-Riitta | Stricker, Bruno H Ch | Elliott, Paul | O’Connor, George T | Strachan, David P | London, Stephanie J | Hall, Ian P | Gudnason, Vilmundur | Tobin, Martin D
Nature Genetics  2011;43(11):1082-1090.
Pulmonary function measures reflect respiratory health and predict mortality, and are used in the diagnosis of chronic obstructive pulmonary disease (COPD). We tested genome-wide association with the forced expiratory volume in 1 second (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) in 48,201 individuals of European ancestry, with follow-up of top associations in up to an additional 46,411 individuals. We identified new regions showing association (combined P<5×10−8) with pulmonary function, in or near MFAP2, TGFB2, HDAC4, RARB, MECOM (EVI1), SPATA9, ARMC2, NCR3, ZKSCAN3, CDC123, C10orf11, LRP1, CCDC38, MMP15, CFDP1, and KCNE2. Identification of these 16 new loci may provide insight into the molecular mechanisms regulating pulmonary function and into molecular targets for future therapy to alleviate reduced lung function.
PMCID: PMC3267376  PMID: 21946350
13.  A Discovery Funnel for Nucleic Acid Binding Drug Candidates 
Drug development research  2011;72(2):178-186.
Computational approaches are becoming increasingly popular for the discovery of drug candidates against a target of interest. Proteins have historically been the primary targets of many virtual screening efforts. While in silico screens targeting proteins has proven successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many non-cannonical DNA structures such as G-quadruplexes, increased efforts are underway to discover new small molecules that can bind selectively to DNA structures. Here, we describe efforts to build an integrated in silico and in vitro platform for discovering compounds that may bind to a chosen DNA target. Millions of compounds are initially screened in silico for selective binding to a particular structure and ranked to identify several hundred best hits. An important element of our strategy is the inclusion of an array of possible competing structures in the in silico screen. The best hundred or so hits are validated experimentally for binding to the actual target structure by a high-throughput 96-well thermal denaturation assay to yield the top ten candidates. Finally, these most promising candidates are thoroughly characterized for binding to their DNA target by rigorous biophysical methods, including isothermal titration calorimetry, differential scanning calorimetry, spectroscopy and competition dialysis.This platform was validated using quadruplex DNA as a target and a newly discovered quadruplex binding compound with possible anti-cancer activity was discovered. Some considerations when embarking on virtual screening and in silico experiments are also discussed.
PMCID: PMC3090163  PMID: 21566705
drug discovery; in silico screening; SURFLEX-DOCK; DNA; G-quadruplex; high-throughput screening
14.  Th2 Cytokine Levels Distort the Association of IL-10 and IFN-γ with Allergic Phenotypes 
ISRN Allergy  2011;2011:405813.
The expression of allergic phenotypes involves complex inter-relationships among several Th2 and Th1 cytokines as well as the regulator cytokine interleukin (IL)-10. These direct or indirect interrelationships may distort the true associations of cytokine responses with these phenotypes. In this study, we aimed to clarify the effects of the regulatory cytokine IL-10 and Th1 cytokine interferon-gamma (IFN-γ) on allergic phenotypes after adjusting for the correlations with Th2 cytokines. After adjusting for Th2 cytokines, IL-10 and IFN-γ were protective against atopy. Adjusted levels of IL-10 and IFN-γ stimulated with house-dust mite (HDM) were significantly lower in atopics than non-atopics, for IL-10 adjusting for IL-5 (P = 0.002), IL-13 (P = 0.012), IL-9 (P = 0.016), and IL-4 (P = 0.043), and for IFN-γ adjusting for IL-5 (P = 0.005), IL-13 (P = 0.005), and IL-9 (P = 0.037). IL-10 and IFN-γ levels stimulated with phytohaemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) exhibited a similar pattern. The adjusted levels of IL-10 and IFN-γ stimulated with HDM, PHA or SEB were all significantly negatively correlated with total serum IgE, except for IFN-γ stimulated with SEB. Levels of Th2 cytokines distort the associations of IL-10 and IFN-γ with allergic phenotypes. Removing the covariance with Th2 cytokines, both IL-10 and IFN-γ were protective against atopy.
PMCID: PMC3658504  PMID: 23724228
15.  Does Genetic Regulation of IgE Begin In-Utero? Evidence from TH1/TH2 Gene Polymorphisms and Cord Blood Total IgE 
Elucidation of early life factors is critical to understand the development of allergic diseases, especially those manifesting in early life such as food allergies and atopic dermatitis. Cord blood IgE (CBIgE) is a recognized risk factor for the subsequent development of allergic diseases. In contrast to numerous genetic studies of total serum IgE in children and adults, limited genetic studies on CBIgE have been conducted.
To test the associations between functional or tagging single nucleotide polymorphisms (SNPs) in genes involved in the TH1/TH2 pathway and CBIgE in a large U.S. inner-city birth cohort.
CBIgE, measured by Phadia ImmnunoCAP, was analyzed as a continuous and a binary variable. The association of each SNP with the two outcomes was tested using tobit and logistic regression models, respectively, with adjustment for pertinent covariates, ancestral proportion, and multiple testing. Ethnic heterogeneity and gene-gene interactions were also explored.
Three SNPs (rs1800925, rs2069743 and rs1295686) in the IL13 gene were significantly associated with CBIgE concentration (p≤6×10-4, pFDR<0.05). These SNPs jointly influenced CBIgE in a dose-response manner (ptrend=9×10-8). Significant associations also were observed for SNPs in the IL13RA1 (rs5956080) and STAT6 (rs11172106) genes. Ethnicity-specific genetic effects were observed for SNPs in the IL5 and GATA3 genes. Several gene-gene interactions (including IL13-IL4R and IL13-STAT6 interactions) were detected in relation to CBIgE.
Our data demonstrated that multiple SNPs were individually and jointly associated with CBIgE, with evidence of gene-gene interactions and ethnic heterogeneity. These findings suggest that genetic regulation of IgE may begin in-utero.
PMCID: PMC3020083  PMID: 21050946
Genetic association; candidate gene; cord blood IgE; gene-gene interaction
16.  Structure-based Drug Design: From Nucleic Acid to Membrane Protein Targets 
The in silico methods for drug discovery are becoming increasingly powerful and useful. That, in combination with increasing computer processor power, in our case using a novel distributed computing grid, has enabled us to greatly enhance our virtual screening efforts. Herein we review some of these efforts using both receptor and ligand-based virtual screening, with the goal of finding new anticancer agents. In particular, nucleic acids are a neglected set of targets, especially the different morphologies of duplex, triplex, and quadruplex DNA, many of which have increasing biological relevance. We also review examples of molecular modeling to understand receptors and using virtual screening against G-protein coupled receptor membrane proteins.
PMCID: PMC3143464  PMID: 19454265
Virtual screening; drug discovery; membrane protein; G-protein coupled receptor; telomere; quadruplex; DNA
17.  Pneumococcal conjugate vaccination at birth in a high-risk setting: No evidence for neonatal T-cell tolerance 
Vaccine  2011;29(33-19):5414-5420.
Concerns about the risk of inducing immune deviation-associated “neonatal tolerance” as described in mice have restricted the widespread adoption of neonatal vaccination. The aim of this study was to demonstrate the immunological feasibility of neonatal pneumococcal conjugate vaccination (PCV) which could potentially protect high-risk infants in resource poor countries against severe pneumococcal disease and mortality in the early critical period of life. Papua New Guinean infants were randomized to be vaccinated with the 7-valent PCV (7vPCV) at birth, 1 and 2 months (neonatal group, n = 104) or at 1, 2 and 3 months of age (infant group, n = 105), or to not receive 7vPCV at all (control group, n = 109). Analysis of vaccine responses at 3 and 9 months of age demonstrated persistently higher type-1 (IFN-γ) and type-2 (IL-5 and IL-13) T-cell responses to the protein carrier CRM197 and IgG antibody titres to 7vPCV serotypes in children vaccinated with 7vPCV according to either schedule as compared to unvaccinated children. In a comprehensive immuno-phenotypic analysis at 9 months of age, no differences in the quantity or quality of vaccine-specific T cell memory responses were found between neonatal vaccinations versus children given their first PCV dose at one month. Hospitalization rates in the first month of life did not differ between children vaccinated with PCV at birth or not. These findings demonstrate that neonatal 7vPCV vaccination is safe and not associated with immunological tolerance. Neonatal immunisation schedules should therefore be considered in high-risk areas where this may result in improved vaccine coverage and the earliest possible protection against pneumococcal disease and death.
PMCID: PMC3146700  PMID: 21645573
Immunisation; Newborn; Pneumococcal conjugate vaccine; Safety; Immunogenicity
18.  Soothing signals: transplacental transmission of resistance to asthma and allergy 
The Journal of Experimental Medicine  2009;206(13):2861-2864.
The progressive rise in the prevalence of allergic diseases since the 1970s is widely attributed to diminished exposure to microbial stimuli, resulting in dysregulated immune functions during early life. Most studies investigating the mechanism behind this phenomenon have focused on postnatal microbial exposure. But emerging evidence suggests that such programming may also occur in the developing fetus as a result of microbial stimulation of the pregnant mother.
PMCID: PMC2806448  PMID: 19995954
19.  Maternal Antibodies to Pneumolysin but Not to Pneumococcal Surface Protein A Delay Early Pneumococcal Carriage in High-Risk Papua New Guinean Infants▿  
Clinical and Vaccine Immunology : CVI  2009;16(11):1633-1638.
Immunization of pregnant women can be an efficient strategy to induce early protection in infants in developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a median age of 19 days. Maternal and cord blood antibody titers to Ply (ρ = 0.824, P < 0.001), PspA1 (ρ = 0.746, P < 0.001), and PspA2 (ρ = 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage (hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95% CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants against pneumococcal disease by reducing carriage risks in both mothers and infants.
PMCID: PMC2772384  PMID: 19776196
20.  Molecular Docking of Intercalators and Groove-Binders to Nucleic Acids Using Autodock and Surflex 
The molecular docking tools Autodock and Surflex accurately reproduce the crystallographic structures of a collection of small molecule ligands that have been shown to bind nucleic acids. Docking studies were performed with the intercalators daunorubicin and ellipticine and the minor groove binders distamycin and pentamidine. Autodock and Surflex dock daunorubicin and distamycin to their nucleic acid targets within a resolution of approximately 2 Å, which is similar to the limit of the crystal structure resolution. However, for the top ranked poses, Autodock and Surflex both dock ellipticine into the correct site but in a different orientation compared to the crystal structure. This appears not only to be partly related to the symmetry of the target nucleic acid, as ellipticine is able to dock from either side of the intercalation site, but also due to the shape of the ligand and docking accuracy. Surflex docks pentamidine in a symmetrically equivalent orientation relative to the crystal structure, while Autodock was able to dock this molecule in the original orientation. In the case of the Surflex docking of pentamidine, the initial rmsd is misleading, given the symmetrical structure of pentamidine. Importantly, the ranking functions of both of these programs are able to return a top pose within approximately 2 Å rmsd for daunorubicin, distamycin, and pentamidine and approximately 3 Å rmsd for ellipticine compared to their respective crystal structures. Some docking challenges and potential pitfalls are explored, such as the importance of hydrogen treatment on ligands as well as the scoring functions of Autodock and Surflex. Overall for this set of complexes, Surflex is preferred over Autodock for virtual screening, as although the results are comparable, Surflex has significantly faster performance and ease of use under the optimal software conditions tested. These experiments show that molecular docking techniques can be successfully extended to include nucleic acid targets, a finding which has important implications for virtual screening applications and in the design of new small molecules to target therapeutically relevant morphologies of nucleic acids.
PMCID: PMC2755229  PMID: 18642866
21.  Neonatal pneumococcal conjugate vaccine immunization primes T cells for preferential Th2 cytokine expression: A randomized controlled trial in Papua New Guinea 
Vaccine  2009;27(9):1340-1347.
The effects of neonatal immunization with 7-valent pneumococcal conjugate vaccine (7vPCV) on development of T-cell memory and general immune maturation were studied in a cohort of Papua New Guinean newborns. Neonatal 7vPCV priming (followed by a dose at 1 and 2 months of age) was associated with enhanced Th2, but not Th1, cytokine responses to CRM197 compared to 7vPCV at 1 and 2 months of age only. T cell responses to non-7vPCV vaccine antigens were similar in all groups, but TLR-mediated IL-6 and IL-10 responses were enhanced in 7vPCV vaccinated compared to controls. Neonatal 7vPCV vaccination primes T cell responses with a polarization towards Th2 with no bystander effects on other T cell responses.
PMCID: PMC2697326  PMID: 19150378
Pneumococcal conjugate vaccine; Neonatal; T cells
22.  Discovery of novel triple helical DNA intercalators by an integrated virtual and actual screening platform 
Nucleic Acids Research  2009;37(4):1280-1287.
Virtual Screening is an increasingly attractive way to discover new small molecules with potential medicinal value. We introduce a novel strategy that integrates use of the molecular docking software Surflex with experimental validation by the method of competition dialysis. This integrated approach was used to identify ligands that selectively bind to the triplex DNA poly(dA)-[poly(dT)]2. A library containing ∼2 million ligands was virtually screened to identify compounds with chemical and structural similarity to a known triplex intercalator, the napthylquinoline MHQ-12. Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12. One of the compounds has a different chemical backbone than MHQ-12, which demonstrates the ability of this strategy to ‘scaffold hop’ and to identify small molecules with novel binding properties. Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity. These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.
PMCID: PMC2651796  PMID: 19136469
23.  Reversal of airway hyperresponsiveness by induction of airway mucosal CD4+CD25+ regulatory T cells 
The Journal of Experimental Medicine  2006;203(12):2649-2660.
An important feature of atopic asthma is the T cell–driven late phase reaction involving transient bronchoconstriction followed by development of airways hyperresponsiveness (AHR). Using a unique rat asthma model we recently showed that the onset and duration of the aeroallergen-induced airway mucosal T cell activation response in sensitized rats is determined by the kinetics of functional maturation of resident airway mucosal dendritic cells (AMDCs) mediated by cognate interactions with CD4+ T helper memory cells. The study below extends these investigations to chronic aeroallergen exposure. We demonstrate that prevention of ensuing cycles of T cell activation and resultant AHR during chronic exposure of sensitized rats to allergen aerosols is mediated by CD4+CD25+Foxp3+LAG3+ CTLA+CD45RC+ T cells which appear in the airway mucosa and regional lymph nodes within 24 h of initiation of exposure, and inhibit subsequent Th-mediated upregulation of AMDC functions. These cells exhibit potent regulatory T (T reg) cell activity in both in vivo and ex vivo assay systems. The maintenance of protective T reg activity is absolutely dependent on continuing allergen stimulation, as interruption of exposure leads to waning of T reg activity and reemergence of sensitivity to aeroallergen exposure manifesting as AMDC/T cell upregulation and resurgence of T helper 2 cytokine expression, airways eosinophilia, and AHR.
PMCID: PMC2118157  PMID: 17088431
24.  Dendritic Cell Immaturity during Infancy Restricts the Capacity To Express Vaccine-Specific T-Cell Memory  
Infection and Immunity  2006;74(2):1106-1112.
The capacity of the immune system in infants to develop stable T-cell memory in response to vaccination is attenuated, and the mechanism(s) underlying this developmental deficiency in humans is poorly understood. The present study focuses on the capacity for expression of in vitro recall responses to tetanus and diphtheria antigens in lymphocytes from 12-month-old infants vaccinated during the first 6 months of life. We demonstrate that supplementation of infant lymphocytes with “matured” dendritic cells (DC) cultured from autologous CD14+ precursors unmasks previously covert cellular immunity in the form of Th2-skewed cytokine production. Supplementation of adult lymphocytes with comparable prematured autologous DC also boosted vaccine-specific T-cell memory expression, but in contrast to the case for the infants, these cytokine responses were heavily Th1 skewed. Compared to adults, infants had significantly fewer circulating myeloid DC (P < 0.0001) and plasmacytoid DC (P < 0.0001) as a proportion of peripheral blood mononuclear cells. These findings suggest that deficiencies in the numbers of antigen-presenting cells and their functional competence at 12 months of age limit the capacity to express effector memory responses and are potentially a key factor in reduced vaccine responsiveness in infants.
PMCID: PMC1360347  PMID: 16428758
25.  Th2-Associated Local Reactions to the Acellular Diphtheria-Tetanus-Pertussis Vaccine in 4- to 6-Year-Old Children  
Infection and Immunity  2005;73(12):8130-8135.
Acellular vaccines against diphtheria-tetanus-pertussis (acellular pertussis) (DTaP) are being progressively introduced into vaccination programs worldwide, with the aim of reducing T-helper 1 (Th1)-associated reactogenicity associated with the cellular diphtheria-tetanus-pertussis (whole-cell pertussis) (DTwP) vaccine. The DTaP vaccine has an improved safety profile in infants, but little information is available concerning the nature of the ensuing immunological memory in older children and how this may affect the reactogenicity of DTaP booster doses. We have addressed this question in the present study by assessing polyclonal and vaccine antigen-specific humoral and cellular immune responses to boosting with DTaP in 4- to 6-year-old children primed during infancy with DTaP (n = 30) or DTwP (n = 16) and by correlating these parameters, in particular cytokine responses, with expression of local side effects at the injection site. Large local reactions (≥50-mm diameter) 24 to 72 h after receiving the DTaP booster occurred in 43% of exclusively DTaP-primed children, in contrast to 6% of children primed with DTwP. These reactions were associated with vigorous T helper 2 (Th2)-polarized memory responses to vaccine antigen exemplified by interleukin 5 (IL-5), IL-6, and IL-13 production and log-scale boosting of tetanus-specific immunoglobulin E and occurred most frequently among children who are intrinsically “high Th2 responders” as detected by in vitro responsiveness to polyclonal mitogen. Our findings suggest that priming during infancy with DTaP promotes stable, boostable Th2-polarized immunity against vaccine antigens, which in a significant subset of children is subsequently associated with local reactions at the booster site. The time course of these reactions suggests that the underlying mechanism involves reactivation of Th2-polarized cellular immune memory.
PMCID: PMC1307058  PMID: 16299307

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