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1.  Epigenomic deconvolution of breast tumors reveals metabolic coupling between constituent cell types 
Cell reports  2016;17(8):2075-2086.
Cancer progression depends on both cell-intrinsic processes and interactions between different cell types. However, large scale assessment of cell type composition and molecular profiles of individual cell types within tumors remains challenging. To address this, we developed Epigenomic Deconvolution (EDec), an in silico method that infers cell type composition of complex tissues as well as DNA methylation and gene transcription profiles of constituent cell types. By applying EDec to The Cancer Genome Atlas (TCGA) breast tumors we detect changes in immune cell infiltration related to patient prognosis, and a striking change in stromal fibroblast to adipocyte ratio across breast cancer subtypes. We further show that a less adipose stroma tends to display lower levels of mitochondrial activity and to be associated with cancerous cells with higher levels of oxidative metabolism. These findings highlight the role of stromal composition in the metabolic coupling between distinct cell types within tumors.
eTOC blurb
Onuchic et al. develop an in silico deconvolution technique (EDec) that can accurately estimate cell type composition and molecular profiles of constituent cell types in the context of breast tumors. Application to breast cancers from TCGA data reveals association between stromal composition and the metabolic phonotype of breast tumors. Explore consortium data at the Cell Press IHEC webportal at
PMCID: PMC5115176  PMID: 27851969
2.  A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity 
The AAPS Journal  2015;17(6):1417-1426.
Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.
PMCID: PMC4627444  PMID: 26205082
ADCC; benralizumab; cell-based assay; mechanism of action; neutralizing antibody
3.  Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays 
Journal of Immunology Research  2016;2016:1485615.
Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.
PMCID: PMC4958433  PMID: 27478855
4.  Pregnancy Outcomes in Autosomal Dominant Polycystic Kidney Disease: a Case-Control Study 
To determine whether autosomal dominant polycystic kidney disease (ADPKD) is associated with adverse fetal outcomes and maternal complications
We identified a cohort of 146 patients seen for pregnancy and cystic kidney disease at Mayo Clinic from 1975 to 2010. From this cohort, 54 patients met the ultrasound diagnostic criteria for ADPKD (ADPKD group), while the other 92 patients were diagnosed as “Simple Cyst” (control group). We compared the fetal and maternal outcomes of pregnancy and long-term maternal prognoses between these two groups.
Overall, the fetal complication rates were similar between the ADPKD and control groups. Rates of spontaneous abortion (15.1% vs. 14%, P=.77) and premature birth (11.1% vs. 6.8%, P=.44) were comparable between groups, while the rate of fetal distress (3.4% vs. 0.7%, P<.01) was increased in the ADPKD group. The rate of preeclampsia in the patients with simple cysts (2%) was similar to that of the general population. In contrast, the pregnant ADPKD patients had higher risks for hypertension, proteinuria, edema, urinary tract infection, renal dysfunction, and preeclampsia during their pregnancies.
ADPKD is associated with increased maternal complications during pregnancy, but only has a slight potential of increased rates of fetal complications.
PMCID: PMC4830432  PMID: 25754208
ADPKD; fetal outcomes; maternal complications; simple cyst disease
6.  Effect of Roux-en-Y Gastric Bypass Surgery on Bile Acid Metabolism in Normal and Obese Diabetic Rats 
PLoS ONE  2015;10(3):e0122273.
In addition to classic functions of facilitating hepatobiliary secretion and intestinal absorption of lipophilic nutrients, bile acids (BA) are also endocrine factors and regulate glucose and lipid metabolism. Recent data indicate that antiobesity bariatric procedures e.g. Roux-en-Y gastric bypass surgery (RYGB), which also remit diabetes, increase plasma BAs in humans, leading to the hypothesis that BAs may play a role in diabetes resolution following surgery. To investigate the effect of RYGB on BA physiology and its relationship with glucose homeostasis, we undertook RYGB and SHAM surgery in Zucker diabetic fatty (ZDF) and normoglycemic Sprague Dawley (SD) rats and measured plasma and fecal BA levels, as well as plasma glucose, insulin, Glucagon like peptide 1 (GLP-1) and Peptide YY (PYY), 2 days before and 3, 7, 14 and 28 days after surgery. RYGB decreased body weight and increased plasma GLP-1 in both SD and ZDF rats while decreasing plasma insulin and glucose in ZDF rats starting from the first week. Compared to SHAM groups, both SD-RYGB and ZDF-RYGB groups started to have increases in plasma total BAs in the second week, which might not contribute to early post-surgery metabolic changes. While there was no significant difference in fecal BA excretion between SD-RYGB and SD-SHAM groups, the ZDF-RYGB group had a transient 4.2-fold increase (P<0.001) in 24-hour fecal BA excretion on post-operative day 3 compared to ZDF-SHAM, which paralleled a significant increase in plasma PYY. Ratios of plasma and fecal cholic acid/chenodeoxycholic acid derived BAs were decreased in RYGB groups. In addition, tissue mRNA expression analysis suggested early intestinal BA reabsorption and potentially reduced hepatic cholic acid production in RYGB groups. In summary, we present novel data on RYGB-mediated changes in BA metabolism to further understand the role of BAs in RYGB-induced metabolic effects in humans.
PMCID: PMC4370587  PMID: 25798945
8.  Genome-wide association analysis identifies six new loci associated with forced vital capacity 
Loth, Daan W. | Artigas, María Soler | Gharib, Sina A. | Wain, Louise V. | Franceschini, Nora | Koch, Beate | Pottinger, Tess | Smith, Albert Vernon | Duan, Qing | Oldmeadow, Chris | Lee, Mi Kyeong | Strachan, David P. | James, Alan L. | Huffman, Jennifer E. | Vitart, Veronique | Ramasamy, Adaikalavan | Wareham, Nicholas J. | Kaprio, Jaakko | Wang, Xin-Qun | Trochet, Holly | Kähönen, Mika | Flexeder, Claudia | Albrecht, Eva | Lopez, Lorna M. | de Jong, Kim | Thyagarajan, Bharat | Alves, Alexessander Couto | Enroth, Stefan | Omenaas, Ernst | Joshi, Peter K. | Fall, Tove | Viňuela, Ana | Launer, Lenore J. | Loehr, Laura R. | Fornage, Myriam | Li, Guo | Wilk, Jemma B. | Tang, Wenbo | Manichaikul, Ani | Lahousse, Lies | Harris, Tamara B. | North, Kari E. | Rudnicka, Alicja R. | Hui, Jennie | Gu, Xiangjun | Lumley, Thomas | Wright, Alan F. | Hastie, Nicholas D. | Campbell, Susan | Kumar, Rajesh | Pin, Isabelle | Scott, Robert A. | Pietiläinen, Kirsi H. | Surakka, Ida | Liu, Yongmei | Holliday, Elizabeth G. | Schulz, Holger | Heinrich, Joachim | Davies, Gail | Vonk, Judith M. | Wojczynski, Mary | Pouta, Anneli | Johansson, Åsa | Wild, Sarah H. | Ingelsson, Erik | Rivadeneira, Fernando | Völzke, Henry | Hysi, Pirro G. | Eiriksdottir, Gudny | Morrison, Alanna C. | Rotter, Jerome I. | Gao, Wei | Postma, Dirkje S. | White, Wendy B. | Rich, Stephen S. | Hofman, Albert | Aspelund, Thor | Couper, David | Smith, Lewis J. | Psaty, Bruce M. | Lohman, Kurt | Burchard, Esteban G. | Uitterlinden, André G. | Garcia, Melissa | Joubert, Bonnie R. | McArdle, Wendy L. | Musk, A. Bill | Hansel, Nadia | Heckbert, Susan R. | Zgaga, Lina | van Meurs, Joyce B.J. | Navarro, Pau | Rudan, Igor | Oh, Yeon-Mok | Redline, Susan | Jarvis, Deborah | Zhao, Jing Hua | Rantanen, Taina | O’Connor, George T. | Ripatti, Samuli | Scott, Rodney J. | Karrasch, Stefan | Grallert, Harald | Gaddis, Nathan C. | Starr, John M. | Wijmenga, Cisca | Minster, Ryan L. | Lederer, David J. | Pekkanen, Juha | Gyllensten, Ulf | Campbell, Harry | Morris, Andrew P. | Gläser, Sven | Hammond, Christopher J. | Burkart, Kristin M. | Beilby, John | Kritchevsky, Stephen B. | Gudnason, Vilmundur | Hancock, Dana B. | Williams, O. Dale | Polasek, Ozren | Zemunik, Tatijana | Kolcic, Ivana | Petrini, Marcy F. | Wjst, Matthias | Kim, Woo Jin | Porteous, David J. | Scotland, Generation | Smith, Blair H. | Viljanen, Anne | Heliövaara, Markku | Attia, John R. | Sayers, Ian | Hampel, Regina | Gieger, Christian | Deary, Ian J. | Boezen, H. Marike | Newman, Anne | Jarvelin, Marjo-Riitta | Wilson, James F. | Lind, Lars | Stricker, Bruno H. | Teumer, Alexander | Spector, Timothy D. | Melén, Erik | Peters, Marjolein J. | Lange, Leslie A. | Barr, R. Graham | Bracke, Ken R. | Verhamme, Fien M. | Sung, Joohon | Hiemstra, Pieter S. | Cassano, Patricia A. | Sood, Akshay | Hayward, Caroline | Dupuis, Josée | Hall, Ian P. | Brusselle, Guy G. | Tobin, Martin D. | London, Stephanie J.
Nature genetics  2014;46(7):669-677.
Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10−8) with FVC in or near EFEMP1, BMP6, MIR-129-2/HSD17B12, PRDM11, WWOX, and KCNJ2. Two (GSTCD and PTCH1) loci previously associated with spirometric measures were related to FVC. Newly implicated regions were followed-up in samples of African American, Korean, Chinese, and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and pathogenesis of restrictive lung disease.
PMCID: PMC4140093  PMID: 24929828
9.  Hypertension in pregnancy is associated with elevated homocysteine levels later in life 
American journal of obstetrics and gynecology  2013;209(5):10.1016/j.ajog.2013.06.030.
Hyperhomocysteinemia is associated with an elevated cardiovascular disease risk. We examined whether women with a history of hypertension in pregnancy are more likely to have a high level of serum homocysteine decades after pregnancy.
Serum homocysteine was measured at a mean age of 60 years in nulliparous women (n = 216), and women with a history of normotensive (n = 1825) or hypertensive (n = 401) pregnancies who participated in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. Relationships between homocysteine and pregnancy history were examined by linear and logistic regression, controlling for multiple covariates including personal and family history of hypertension, diabetes, obesity, tobacco use, and demographics.
A history of hypertension in pregnancy, when compared with normotensive pregnancy, was associated with a 4.5% higher serum homocysteine level (P = .015) and 1.60-fold increased odds of having an elevated homocysteine (95% confidence interval, 1.15–2.21; P = .005) after adjusting for potentially confounding covariates. In contrast, a history of normotensive pregnancy, as compared with nulliparity, was associated with a 6.1% lower serum homocysteine level (P = .005) and a 0.49-fold reduced odds of elevated homocysteine levels (95% confidence interval, 0.32–0.74; P < .001).
Homocysteine levels decades after pregnancy are higher in women with a history of pregnancy hypertension, even after controlling for potential confounders. Thus, pregnancy history may prompt homocysteine assessment and risk modification in an attempt at primary prevention of cardiovascular disease.
PMCID: PMC3825794  PMID: 23791689
biomarker; cardiovascular disease; homocysteine; hypertension in pregnancy; preeclampsia
10.  The Treatment of Hypertension During Pregnancy: When Should Blood Pressure Medications Be Started? 
Current cardiology reports  2013;15(11):10.1007/s11886-013-0412-0.
Hypertensive pregnancy disorders (HPD) are important causes of maternal and fetal morbidity and mortality worldwide. In addition, a history of HPD has been associated with an increased risk for maternal cardiovascular disease later in life, possibly due to irreversible vascular and metabolic changes that persist beyond the affected pregnancies. Therefore, treatment of HPD may not only improve immediate pregnancy outcomes, but also the maternal long-term cardiovascular health. Unlike the recommendations for hypertension treatment in the general population, treatment recommendations of HPD have not changed substantially for more than two decades. This is particularly true for mild to moderate hypertension in pregnancy, defined as a blood pressure of 140–159/90–109 mm Hg.
This review focuses on the goals of therapy, treatment strategies, and new developments in the field of HPD that should be taken into account when considering blood pressure targets and pharmacological options for treatment of hypertension in pregnant women.
PMCID: PMC3859314  PMID: 24057769
Hypertension; Treatment; Pregnancy induced; Antihypertensive agents; Cardiovascular diseases in women; Blood pressure medications
11.  Replication and fine mapping of asthma-associated loci in individuals of African ancestry 
Human genetics  2013;132(9):1039-1047.
Asthma originates from genetic and environmental factors with about half the risk of disease attributable to heritable causes. Genome-wide association studies, mostly in populations of European ancestry, have identified numerous asthma-associated single nucleotide polymorphisms (SNPs). Studies in populations with diverse ancestries allow both for identification of robust associations that replicate across ethnic groups and for improved resolution of associated loci due to different patterns of linkage disequilibrium between ethnic groups. Here we report on an analysis of 745 African-American subjects with asthma and 3,238 African-American control subjects from the Candidate Gene Association Resource (CARe) Consortium, including analysis of SNPs imputed using 1,000 Genomes reference panels and adjustment for local ancestry. We show strong evidence that variation near RAD50/IL13, implicated in studies of European ancestry individuals, replicates in individuals largely of African ancestry. Fine mapping in African ancestry populations also refined the variants of interest for this association. We also provide strong or nominal evidence of replication at loci near ORMDL3/GSDMB, IL1RLML18R1, and 10pl4, all previously associated with asthma in European or Japanese populations, but not at the PYHIN1 locus previously reported in studies of African-American samples. These results improve the understanding of asthma genetics and further demonstrate the utility of genetic studies in populations other than those of largely European ancestry.
PMCID: PMC3975655  PMID: 23666277
12.  APOM and High-Density Lipoprotein are associated with Lung Function and Percent Emphysema 
The European respiratory journal  2013;43(4):1003-1017.
Chronic obstructive pulmonary disease (COPD) is linked to cardiovascular disease; however, there are few studies on the associations of cardiovascular genes with COPD.
We assessed the association of lung function with 2,100 genes selected for cardiovascular diseases among 20,077 European-Americans and 6,900 African-Americans. We performed replication of significant loci in the other racial group and an independent consortium of Europeans, tested the associations of significant loci with percent emphysema, and examined gene expression in an independent sample. We then tested the association of a related lipid biomarker with FEV1/FVC and percent emphysema.
We identified one new polymorphism for FEV1/FVC (rs805301) in European-Americans (p=1.3×10−6) and a second (rs707974) in the combined European-American and African-American analysis (p=1.38×10−7). Both SNPs flank the gene for apolipoprotein M (apoM), a component of HDL. Both replicated in an independent cohort. SNPs in a second gene related to apoM and HDL, PCSK9, were associated with FEV1/FVC among African-Americans. rs707974 was associated with percent emphysema among European-Americans and African-Americans, and APOM expression was related to FEV1/FVC and percent emphysema. Higher HDL levels were associated with lower FEV1/FVC and greater percent emphysema.
These findings suggest a novel role for the APOM/HDL pathway in the pathogenesis of COPD and emphysema.
PMCID: PMC4041087  PMID: 23900982
Apolipoproteins; Cholesterol; Percent Emphysema; Polymorphism, Single Nucleotide; Pulmonary Disease, Chronic Obstructive
13.  Mass spectrometry as a novel method for detection of podocyturia in pre-eclampsia 
Nephrology Dialysis Transplantation  2012;28(6):1555-1561.
Podocyturia, i.e. urinary loss of viable podocytes, may serve as a diagnostic tool for pre-eclampsia and as a marker of active renal disease. The current method to detect podocyturia is technically complex, lengthy and requires a high level of expertise for interpretation. The aim of this study was to develop a new technique for the identification of urinary podocytes, based on the detection of podocyte-specific tryptic peptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), which will provide an operator-independent and highly reproducible method.
Methods and Results
The diagnosis of pre-eclampsia was confirmed in the presence of hypertension (>140/90 mmHg) and proteinuria >0.3 g/24 h urine. The diagnosis of HELLP was confirmed based on the accepted clinical criteria of hemolysis, elevated liver enzymes and low platelet count.
Random urine samples within 24 h prior to delivery were collected and centrifuged. One half of the sediment was cultured for 24 h to select for viable cells and then stained with a podocin antibody, followed by a secondary fluorescein isothiocyanate-labeled antibody to identify podocytes. The second half of the pellet was solubilized, digested and analyzed by LC-MS/MS using an internal standard. We have recruited 13 patients with pre-eclampsia and 6 patients with pre-eclampsia/HELLP syndrome. The presence of podocytes was confirmed in all patients by the podocyte culture method. In the respective samples, the presence of a podocin-specific tryptic peptide was confirmed with LC-MS/MS technology.
The LC-MS/MS method is a reliable technology for the identification of urinary podocytes, based on the presence of podocyte-specific proteins in the urine.
PMCID: PMC3685302  PMID: 22523117
mass spectrometry; podocyturia; pre-eclampsia
Hypertension  2013;61(6):1289-1296.
Podocyturia, the shedding of live podocytes, is present at delivery in women with preeclampsia. The aim of this study was to test whether podocyturia is present earlier in pregnancy and predicts for preeclampsia. We also aimed to compare test characteristics of podocyturia to those of angiogenic factors previously implicated in the pathogenesis of this disorder. We prospectively enrolled 315 women who provided blood and urine samples at the end of the 2nd trimesters of their pregnancies (median 27 gestational weeks) and within 24 hours of their deliveries (median 39.5 gestational weeks). Blood samples were analyzed for angiogenic markers, including placental growth factor, the soluble receptor fms-like tyrosine kinase receptor-1 for vascular endothelial growth factor, and endoglin. The urine sediments were analyzed for podocytes, identified by staining for podocin after culturing the urinary sediments for 24 hours. This analysis included all women who developed preeclampsia (n=15), gestational hypertension (n=15), and a subsample of women who remained normotensive throughout pregnancy (n=44), matched for maternal age and number of previous pregnancies, to those who developed preeclampsia. At the second trimester collection, all women who developed preeclampsia had podocyturia, compared to none of those who remained normotensive or were diagnosed with gestational hypertension. Podocyturia in the second trimester had a significantly greater sensitivity and specificity for the subsequent diagnosis of preeclampsia than any single angiogenic marker, or a combination thereof. Screening for podocyturia at the end of the second trimester may allow for accurate identification of pregnant women at risk for preeclampsia.
PMCID: PMC3713793  PMID: 23529165
podocyturia; proteinuria; preeclampsia; pregnancy hypertension; high risk pregnancy
15.  Persistent Urinary Podocyte Loss following Preeclampsia May Reflect Subclinical Renal Injury 
PLoS ONE  2014;9(3):e92693.
Studies have shown that podocyturia, i.e., urinary loss of viable podocytes (glomerular epithelial cells), is associated with proteinuria in preeclampsia. We postulated that urinary podocyte loss may persist after preeclamptic pregnancies, thus resulting in renal injury. This may lead to future chronic renal injury. In addition, we compared the postpartum levels of the angiogenic factors, which previously have been associated with preeclampsia, between normotensive versus preeclamptic pregnancies.
Study Design
The diagnosis of preeclampsia was confirmed using standard clinical criteria. Random blood and urine samples were obtained within 24 hours prior to delivery and 5 to 8 weeks postpartum. Urine sediments were cultured for 24 hours to select for viable cells and staining for podocin was used to identify podocytes. Serum samples were analyzed for the levels of angiogenic markers using ELISA (enzyme-linked immunosorbent assay) methodology.
At delivery, preeclamptic patients (n = 10) had significantly higher proteinuria (p = 0.006) and podocyturia (p<0.001) than normotensive pregnant patients (n = 18). Postpartum proteinuria was similar between these two groups (p = 0.37), while podocyturia was present in 3 of 10 women with preeclampsia and in none of the normotensive controls (p = 0.037). Angiogenic marker levels, including placental growth factor, soluble vascular endothelial growth factor receptor fms-like tyrosine kinase receptor-1 and endoglin, were not significantly different between women with preeclampsia and women with a normotensive pregnancy, either at delivery or postpartum.
Persistent urinary podocyte loss after preeclamptic pregnancies may constitute a marker of ongoing, subclinical renal injury.
PMCID: PMC3963957  PMID: 24664365
16.  Joint Effects of Smoking and Sedentary Lifestyle on Lung Function in African Americans: The Jackson Heart Study Cohort 
This study examined: (a) differences in lung function between current and non current smokers who had sedentary lifestyles and non sedentary lifestyles and (b) the mediating effect of sedentary lifestyle on the association between smoking and lung function in African Americans. Sedentary lifestyle was defined as the lowest quartile of the total physical activity score. The results of linear and logistic regression analyses revealed that non smokers with non sedentary lifestyles had the highest level of lung function, and smokers with sedentary lifestyles had the lowest level. The female non-smokers with sedentary lifestyles had a significantly higher FEV1% predicted and FVC% predicted than smokers with non sedentary lifestyles (93.3% vs. 88.6%; p = 0.0102 and 92.1% vs. 86.9%; p = 0.0055 respectively). FEV1/FVC ratio for men was higher in non smokers with sedentary lifestyles than in smokers with non sedentary lifestyles (80.9 vs. 78.1; p = 0.0048). Though smoking is inversely associated with lung function, it seems to have a more deleterious effect than sedentary lifestyle on lung function. Physically active smokers had higher lung function than their non physically active counterparts.
PMCID: PMC3945550  PMID: 24477212
lung function; Jackson Heart Study; African Americans; smoking; sedentary lifestyle
17.  Suppression of soluble T cell-associated proteins by an anti-interferon-α monoclonal antibody in adult patients with dermatomyositis or polymyositis 
Rheumatology (Oxford, England)  2013;53(4):686-695.
Objective. The aim of this study was to identify serum markers that are modulated by an investigational anti-IFN-α mAb, sifalimumab, in adult DM or PM patients.
Methods. In a phase 1b clinical trial, sera were collected from a total of 48 DM or PM adult patients receiving either placebo for 3 months or sifalimumab for 6 months. Samples were tested for 128 selected proteins using a multiplex luminex immunoassay. Muscle biopsies from selected patients were stained for T cell infiltration using an anti-CD3 antibody.
Results. A robust overexpression of multiple serum proteins in DM or PM patients was observed, particularly in patients with an elevated baseline type I IFN gene signature in the blood or muscle. Neutralization of the type I IFN gene signature by sifalimumab resulted in coordinated suppression of T cell-related proteins such as soluble IL-2RA, TNF receptor 2 (TNFR2) and IL-18. Muscle biopsies from two patients with the highest serum protein suppression were selected and found to have a pronounced reduction of muscle T cell infiltration. Down-regulation of IL-2RA correlated with favourable manual muscle test 8 (MMT-8) alterations in sifalimumab-dosed patients.
Conclusion. A reduced level of multiple T cell-associated proteins after sifalimumab but not placebo administration suggests a suppressive effect of blocking type I IFN signalling on T cell activation and chemoattraction that may lead to a reduction of T cell infiltration in the muscle of myositis patients. Further, soluble IL-2RA changes from baseline may serve as a responsive and/or predictive marker for type I IFN-targeted therapy in adult DM or PM patients.
PMCID: PMC3970566  PMID: 24357810
dermatomyositis; polymyositis; type I interferon; soluble interleukin-2 receptor; T cell infiltration
18.  Long QT Syndrome–Associated Mutations in Intrauterine Fetal Death 
JAMA : the journal of the American Medical Association  2013;309(14):10.1001/jama.2013.3219.
Intrauterine fetal death or stillbirth occurs in approximately 1 out of every 160 pregnancies and accounts for 50% of all perinatal deaths. Postmortem evaluation fails to elucidate an underlying cause in many cases. Long QT syndrome (LQTS) may contribute to this problem.
To determine the spectrum and prevalence of mutations in the 3 most common LQTS susceptible genes (KCNQ1, KCNH2, and SCN5A) for a cohort of unexplained cases.
Design, Setting, and Patients
In this case series, retrospective postmortem genetic testing was conducted on a convenience sample of 91 unexplained intrauterine fetal deaths (mean [SD] estimated gestational age at fetal death, 26.3 [8.7] weeks) that were collected from 2006-2012 by the Mayo Clinic, Rochester, Minnesota, or the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. More than 1300 ostensibly healthy individuals served as controls. In addition, publicly available exome databases were assessed for the general population frequency of identified genetic variants.
Main Outcomes and Measures
Comprehensive mutational analyses of KCNQ1 (KV7.1, LQTS type 1), KCNH2 (HERG/KV11.1, LQTS type 2), and SCN5A (NaV1.5, LQTS type 3) were performed using denaturing high-performance liquid chromatography and direct DNA sequencing on genomic DNA extracted from decedent tissue. Functional analyses of novel mutations were performed using heterologous expression and patch-clamp recording.
The 3 putative LQTS susceptibility missense mutations (KCNQ1, p.A283T; KCNQ1, p.R397W; and KCNH2[1b], p.R25W), with a heterozygous frequency of less than 0.05% in more than 10000 publicly available exomes and absent in more than 1000 ethnically similar control patients, were discovered in 3 intrauterine fetal deaths (3.3% [95% CI, 0.68%-9.3%]). Both KV7.1-A283T (16-week male) and KV7.1-R397W (16-week female) mutations were associated with marked KV7.1 loss-of-function consistent with in utero LQTS type 1, whereas the HERG1b-R25W mutation (33.2-week male) exhibited a loss of function consistent with in utero LQTS type 2. In addition, 5 intrauterine fetal deaths hosted SCN5A rare nonsynonymous genetic variants (p.T220I, p.R1193Q, involving 2 cases, and p.P2006A, involving 2 cases) that conferred in vitro electrophysiological characteristics consistent with potentially proarrhythmic phenotypes.
Conclusions and Relevance
In this molecular genetic evaluation of 91 cases of intrauterine fetal death, missense mutations associated with LQTS susceptibility were discovered in 3 cases (3.3%) and overall, genetic variants leading to dysfunctional LQTS-associated ion channels in vitro were discovered in 8 cases (8.8%). These preliminary findings may provide insights into mechanisms of some cases of stillbirth.
PMCID: PMC3852902  PMID: 23571586
19.  Genetic architecture controlling variation in grain carotenoid composition and concentrations in two maize populations 
Key message
Genetic control of maize grain carotenoid profiles is coordinated through several loci distributed throughout three secondary metabolic pathways, most of which exhibit additive, and more importantly, pleiotropic effects.
The genetic basis for the variation in maize grain carotenoid concentrations was investigated in two F2:3 populations, DEexp × CI7 and A619 × SC55, derived from high total carotenoid and high β-carotene inbred lines. A comparison of grain carotenoid concentrations from population DEexp × CI7 grown in different environments revealed significantly higher concentrations and greater trait variation in samples harvested from a subtropical environment relative to those from a temperate environment. Genotype by environment interactions was significant for most carotenoid traits. Using phenotypic data in additive, environment-specific genetic models, quantitative trait loci (QTL) were identified for absolute and derived carotenoid traits in each population, including those specific to the isomerization of β-carotene. A multivariate approach for these correlated traits was taken, using carotenoid trait principal components (PCs) that jointly accounted for 97 % or more of trait variation. Component loadings for carotenoid PCs were interpreted in the context of known substrate-product relationships within the carotenoid pathway. Importantly, QTL for univariate and multivariate traits were found to cluster in close proximity to map locations of loci involved in methyl-erythritol, isoprenoid and carotenoid metabolism. Several of these genes, including lycopene epsilon cyclase, carotenoid cleavage dioxygenase1 and beta-carotene hydroxylase, were mapped in the segregating populations. These loci exhibited pleiotropic effects on α-branch carotenoids, total carotenoid profile and β-branch carotenoids, respectively. Our results confirm that several QTL are involved in the modification of carotenoid profiles, and suggest genetic targets that could be used for the improvement of total carotenoid and β-carotene in future breeding populations.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-013-2179-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3825500  PMID: 24042570
20.  Normal early pregnancy 
Epigenetics  2012;7(7):729-734.
The objective of this study was to analyze genome-wide differential methylation patterns in maternal leukocyte DNA in early pregnant and non-pregnant states. This is an age and body mass index matched case-control study comparing the methylation patterns of 27,578 cytosine-guanine (CpG) sites in 14,495 genes in maternal leukocyte DNA in early pregnancy (n = 14), in the same women postpartum (n = 14), and in nulligravid women (n = 14) on a BeadChip platform. Transient widespread hypomethylation was found in early pregnancy as compared with the non-pregnant states. Methylation of nine genes was significantly different in early pregnancy compared with both postpartum and nulligravid states (< 10% False Discovery Rate). Early pregnancy may be characterized by widespread hypomethylation compared with non-pregnant states; there is no apparent permanent methylation imprint after a normal term gestation. Nine potential candidate genes were identified as differentially methylated in early pregnancy and may play a role in the maternal adaptation to pregnancy.
PMCID: PMC3414393  PMID: 22647708
DNA Methylation; epigenetics; immune; leukocyte; maternal; postpartum; pregnancy
21.  Acute Interstitial Nephritis Proteinuria and Herpes Simplex Virus Hepatitis in Pregnancy Mimic HELLP Syndrome (Hemolysis, Elevated Liver Enzymes, Low Platelets) 
AJP Reports  2011;1(2):115-118.
Elevated transaminases, hemolysis, and thrombocytopenia in pregnancy are most often caused by a preeclampsia variant—HELLP syndrome (hemolysis, elevated liver enzymes, low platelets). In atypical cases, it is important to consider other causes, such as herpes simplex virus (HSV) hepatitis. Acute interstitial nephritis (AIN)-induced proteinuria can make distinguishing HELLP from its mimics more difficult. A 43-year-old G4P3 gestational carrier at 28 weeks had abnormal laboratory findings consistent with HELLP, including proteinuria. However, she was normotensive and febrile, prompting an investigation into other possible causes of her signs and symptoms. She ultimately was diagnosed with disseminated HSV infection, started on definitive therapy, and allowed to continue her pregnancy to term. The proteinuria was attributed to AIN. AIN can cause proteinuria in the critically ill pregnant patient. When mimics of HELLP syndrome, such as disseminated HSV infection, are the cause of critical illness, the presence of AIN-induced proteinuria may falsely implicate a hypertensive disorder of pregnancy, resulting in iatrogenic premature delivery of the fetus and failure to initiate definitive potential lifesaving treatment.
PMCID: PMC3653532  PMID: 23705099
Herpes simplex virus; hepatitis; proteinuria; acute interstitial nephritis; HELLP
22.  Diabetes, Impaired Glucose Tolerance, and Metabolic Biomarkers in Individuals with Normal Glucose Tolerance are Inversely Associated with Lung Function: The Jackson Heart Study 
Lung  2011;189(4):311-321.
The objectives of this study were to test the hypothesis that diabetes and impaired glucose tolerance (IGT), diabetes control and diabetes duration, and metabolic biomarkers in adults with normal glucose tolerance (NGT) are inversely associated with spirometry-measured lung function. We conducted a cross-sectional observational cohort study that included nonsmoking African American adults (n = 2,945; mean age = 52.5 ± 12.6 years; 69.2% female), who were free of cardiovascular disease, from the Jackson Heart Study. The interventions were diabetes, metabolic biomarkers and lung function. We measured the associations of glycemia with forced expiratory volume (FEV) in 1 s, FEV in 6 s, and vital capacity. Multivariable adjusted mean lung function values were lower among adults with diabetes and IGT (in women only, but not after adjustment for waist circumference) than adults with NGT. Among adults with diabetes, no associations were observed between lung function and diabetes control or duration. In women with NGT, lower lung function was consistently associated with higher glucose levels and less consistently with higher insulin levels and insulin resistance. Lower lung function was consistently associated with higher insulin levels and insulin resistance and less consistently associated with insulin and hemoglobin A1c in men with NGT. Overall, our findings generally support the hypothesis that diabetes, IGT, and increased levels of metabolic biomarkers in individuals with NGT are inversely associated with lung function in African Americans, independent of adiposity.
PMCID: PMC3209803  PMID: 21567165
Forced expiratory volume in 1 s; Insulin resistance; Spirometry; Vital capacity; Impaired glucose tolerance
23.  Posterior Reversible Encephalopathy Syndrome and Eclampsia: Pressing the Case for More Aggressive Blood Pressure Control 
Mayo Clinic Proceedings  2011;86(9):851-856.
OBJECTIVE: To assess the prevalence, clinical presentations, and neuroimaging abnormalities in a series of patients treated for eclampsia at Mayo Clinic in Rochester, MN.
PATIENTS AND METHODS: We reviewed the records of all pregnant patients diagnosed as having eclampsia at Mayo Clinic in Rochester, MN, between January 1, 2001, and December 31, 2008. All patients who underwent neuroimaging were identified, and all studies were reviewed by an independent neuroradiologist. Comparisons were made between groups who did and did not undergo imaging to identify differentiating clinical or laboratory variables.
RESULTS: Thirteen cases of eclampsia were found, with neuroimaging studies available for 7: magnetic resonance imaging (n=6) and computed tomography (n=1). All 7 patients developed eclamptic seizures, and 2 of 7 patients had severe hypertension, with recorded systolic blood pressures exceeding 180 mm Hg. Neuroimaging showed characteristic changes of posterior reversible encephalopathy syndrome (PRES) in all patients. Follow-up imaging showed resolution in 2 of 3 patients; 1 patient had residual neuroimaging abnormalities.
CONCLUSION: Our results suggest that the clinical syndrome of eclampsia is associated with an anatomical substrate that is recognizable by neuroimaging as PRES. The levels of blood pressure elevation are lower than those reported in cases of PRES because of hypertensive encephalopathy. Further studies are needed to determine whether more aggressive blood pressure control and early neuroimaging may have a role in the management of these patients.
PMCID: PMC3258001  PMID: 21878596
24.  Batch effect correction for genome-wide methylation data with Illumina Infinium platform 
BMC Medical Genomics  2011;4:84.
Genome-wide methylation profiling has led to more comprehensive insights into gene regulation mechanisms and potential therapeutic targets. Illumina Human Methylation BeadChip is one of the most commonly used genome-wide methylation platforms. Similar to other microarray experiments, methylation data is susceptible to various technical artifacts, particularly batch effects. To date, little attention has been given to issues related to normalization and batch effect correction for this kind of data.
We evaluated three common normalization approaches and investigated their performance in batch effect removal using three datasets with different degrees of batch effects generated from HumanMethylation27 platform: quantile normalization at average β value (QNβ); two step quantile normalization at probe signals implemented in "lumi" package of R (lumi); and quantile normalization of A and B signal separately (ABnorm). Subsequent Empirical Bayes (EB) batch adjustment was also evaluated.
Each normalization could remove a portion of batch effects and their effectiveness differed depending on the severity of batch effects in a dataset. For the dataset with minor batch effects (Dataset 1), normalization alone appeared adequate and "lumi" showed the best performance. However, all methods left substantial batch effects intact in the datasets with obvious batch effects and further correction was necessary. Without any correction, 50 and 66 percent of CpGs were associated with batch effects in Dataset 2 and 3, respectively. After QNβ, lumi or ABnorm, the number of CpGs associated with batch effects were reduced to 24, 32, and 26 percent for Dataset 2; and 37, 46, and 35 percent for Dataset 3, respectively. Additional EB correction effectively removed such remaining non-biological effects. More importantly, the two-step procedure almost tripled the numbers of CpGs associated with the outcome of interest for the two datasets.
Genome-wide methylation data from Infinium Methylation BeadChip can be susceptible to batch effects with profound impacts on downstream analyses and conclusions. Normalization can reduce part but not all batch effects. EB correction along with normalization is recommended for effective batch effect removal.
PMCID: PMC3265417  PMID: 22171553
25.  Safety profile and clinical activity of multiple subcutaneous doses of MEDI-528, a humanized anti-interleukin-9 monoclonal antibody, in two randomized phase 2a studies in subjects with asthma 
Interleukin-9 (IL-9)-targeted therapies may offer a novel approach for treating asthmatics. Two randomized placebo-controlled studies were conducted to assess the safety profile and potential efficacy of multiple subcutaneous doses of MEDI-528, a humanized anti-IL-9 monoclonal antibody, in asthmatics.
Study 1: adults (18-65 years) with mild asthma received MEDI-528 (0.3, 1, 3 mg/kg) or placebo subcutaneously twice weekly for 4 weeks. Study 2: adults (18-50 years) with stable, mild to moderate asthma and exercise-induced bronchoconstriction received 50 mg MEDI-528 or placebo subcutaneously twice weekly for 4 weeks. Adverse events (AEs), pharmacokinetics (PK), immunogenicity, asthma control (including asthma exacerbations), and exercise challenge test were evaluated in study 1, study 2, or both.
In study 1 (N = 36), MEDI-528 showed linear serum PK; no anti-MEDI-528 antibodies were detected. Asthma control: 1/27 MEDI-528-treated subjects had 1 asthma exacerbation, and 2/9 placebo-treated subjects had a total of 4 asthma exacerbations (one considered a serious AE). In study 2, MEDI-528 (n = 7) elicited a trend in the reduction in mean maximum decrease in FEV1 post-exercise compared to placebo (n = 2) (-6.49% MEDI-528 vs -12.60% placebo; -1.40% vs -20.10%; -5.04% vs -15.20% at study days 28, 56, and 150, respectively). Study 2 was halted prematurely due to a serious AE in an asymptomatic MEDI-528-treated subject who had an abnormal brain magnetic resonance imaging that was found to be an artifact on further evaluation.
In these studies, MEDI-528 showed an acceptable safety profile and findings suggestive of clinical activity that support continued study in subjects with mild to moderate asthma.
Trial registration
ClinicalTrials (NCT): NCT00507130 and ClinicalTrials (NCT): NCT00590720
PMCID: PMC3058114  PMID: 21356110

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