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1.  Changes in HbA1c Level over a 12-Week Follow-up in Patients with Type 2 Diabetes following a Medication Change 
PLoS ONE  2014;9(3):e92458.
Current guidance about the interval needed before retesting HbA1c when monitoring for glycaemic control is based on expert opinion rather than well-powered studies. The aim of our work was to explore how fast HbA1c changes after a change in glucose-lowering medication. This has implications for whether routine HbA1c testing intervals before 12 weeks could inform diabetes medication adjustments.
This 12-week cohort study recruited patients from 18 general practices in the United Kingdom with non-insulin treated diabetes who were initiating or changing dose of oral glucose-lowering medication. HbA1c was measured at baseline and 2, 4, 8 and 12 weeks after recruitment. HbA1c levels at earlier time intervals were correlated with 12-week HbA1c. A ROC curve analysis was used to identify the 8-week threshold above which medication adjustment may be clinically appropriate.
Ninety-three patients were recruited to the study. Seventy-nine patients with no change in medication and full 12-week follow-up had the following baseline characteristics: mean±standard deviation age of 61.3±10.8 years, 34% were female and diabetes duration of 6.0±4.3 years. Mean HbA1c at baseline, 2, 4, 8 and 12 weeks was 8.7±1.5%, (72.0±16.8 mmol/mol) 8.6±1.6% (70.7±17.0 mmol/mol), 8.4±1.5% (68.7±15.9 mmol/mol), 8.2±1.4% (66.3±15.8 mmol/mol) and 8.1±1.4% (64.8±15.7 mmol/mol) respectively. At the end of the study 61% of patients had sub-optimal glycaemic control (HbA1c>7.5% or 59 mmol/mol). The 8-week change correlated significantly with the 12-week change in HbA1c and an HbA1c above 8.2% (66 mmol/mol) at 8 weeks correctly classified all 28 patients who had not achieved glycaemic control by 12 weeks.
This is the first study designed with sufficient power to examine short-term changes in HbA1c. The 12-week change in HbA1c can be predicted 8 weeks after a medication change. Many participants who had not achieved glycaemic control after 12 weeks may have benefitted from an earlier review of their HbA1c and medication.
PMCID: PMC3965408  PMID: 24667212
2.  Associations of Ionizing Radiation and Breast Cancer-Related Serum Hormone and Growth Factor Levels in Cancer-Free Female A-Bomb Survivors 
Radiation research  2011;176(5):678-687.
Levels of exposure to ionizing radiation are increasing for women worldwide due to the widespread use of CT and other radiologic diagnostic modalities. Exposure to ionizing radiation as well as increased levels of estradiol and other sex hormones are acknowledged breast cancer risk factors, but the effects of whole-body radiation on serum hormone levels in cancer-free women are unknown. This study examined whether ionizing radiation exposure is associated with levels of serum hormones and other markers that may mediate radiation-associated breast cancer risk. Serum samples were measured from cancer-free women who attended biennial health examinations with a wide range of past radiation exposure levels (N = 412, ages 26–79). The women were selected as controls for separate case-control studies from a cohort of A-bomb survivors. Outcome measures included serum levels of total estradiol, bioavailable estradiol, testosterone, progesterone, prolactin, insulin-like growth factor-1 (IGF1), insulin-like growth factor-binding protein 3 (IGFBP-3), and ferritin. Relationships were assessed using repeated-measures regression models fitted with generalized estimating equations. Geometric mean serum levels of total estradiol and bioavailable estradiol increased with 1 Gy of radiation dose among samples collected from postmenopausal women (17%1Gy, 95% CI: 1%–36% and 21%1Gy, 95% CI: 4%–40%, respectively), while they decreased in samples collected from premenopausal women (−11%1Gy, 95% CI: −20%–1% and −12%1Gy, 95% CI: −20%– −2%, respectively). Interactions by menopausal status were significant (P = 0.003 and P < 0.001, respectively). Testosterone levels increased with radiation dose in postmenopausal samples (30.0%1Gy, 95% CI: 13%–49%) while they marginally decreased in premenopausal samples (−10%1Gy, 95% CI: −19%–0%) and the interaction by menopausal status was significant (P < 0.001). Serum levels of IGF1 increased linearly with radiation dose (11%1Gy, 95% CI: 2%–18%) and there was a significant interaction by menopausal status (P = 0.014). Radiation-associated changes in serum levels of estradiol, bioavailable estradiol, testosterone and IGF1 were modified by menopausal status at the time of collection. No associations with radiation were observed in serum levels of progesterone, prolactin, IGFBP-3 or ferritin.
PMCID: PMC3925891  PMID: 21718103
3.  Normal ranges of heart rate and respiratory rate in children from birth to 18 years: a systematic review of observational studies 
Lancet  2011;377(9770):1011-1018.
Although heart rate and respiratory rate are routinely measured in children in acute settings, current reference ranges are not evidence-based. The aim of this study is to derive new centile charts for heart rate and respiratory rate using systematic review data from existing studies, and to compare these with existing international ranges.
We searched MEDLINE, EMBASE, and CINAHL to April 2009, and reference lists to identify studies which had measured heart rate and/or respiratory rate in normal children between birth and 18 years of age. We used a non-parametric kernel regression method to create centile charts for heart rate and respiratory rate with respect to age. We compared existing reference ranges with those derived from the centile charts.
We included 69 studies, 59 of which provided data on the heart rate of 143,346 children, and 20 on the respiratory rate of 3,881 children. Our new centile charts demonstrate the decline in respiratory rate from birth to early adolescence, with the steepest decline apparent in infants under two years; decreasing from a median of 44 breaths/minutes at birth to 26 breaths/minute at the age of two. The heart rate centile chart demonstrates a small peak at one month of age. The median heart rate increases from 127 beats/minute at birth to a maximum of 145 beats/minute at approximately one month of age, before decreasing to 113 beats/minute by the age of two. Comparison of the centile charts with existing published reference ranges for heart rate and respiratory rate show marked disagreement with the centile charts, with limits from published ranges frequently exceeding the 99th and 1st centiles, or crossing the median.
Our review shows that existing international guidelines for heart rate and respiratory rate in children are not based on evidence. We have created new centile charts based on a systematic review of studies which have measured these vital signs in normal children. Clinical and resuscitation guidelines should be updated in the light of these evidence-based reference ranges.
Research funded by the National Institute for Health Research programme grant for applied research ‘Development and implementation of new diagnostic processes and technologies in primary care’. SF was funded by the Engineering and Physical Sciences Research Council and the National Institute for Health Research Biomedical Research Centre Programme.
PMCID: PMC3789232  PMID: 21411136
children; heart rate; respiratory rate; normal; centiles; ranges
4.  An unusual cause of hearing loss in an 81-year-old man 
BMJ Case Reports  2011;2011:bcr0520114212.
An 81-year-old man presented to accident and emergency with a recent history of rapidly progressive bilateral hearing loss. Further questioning revealed recent sinusitis, fevers and general malaise. Initial bloods showed raised inflammatory markers, raised creatinine, low albumin and urine dip was positive for blood and protein. Chest x-ray showed faint bilateral mid-zone infiltrates. He was treated with intravenous methylprednisolone (500 mg×3) for presumed Wegener’s granulomatosis which was later confirmed by c-anti-neutrophil cytoplasmic antibodies testing (proteinase-3 positive) and supportive findings on CT chest and sinuses. He was discharged after 10 days in hospital on oral prednisolone and cyclophosphamide. He is continuing treatment and seen regularly in outpatient follow-up. Unfortunately his hearing has not fully recovered and he is awaiting a hearing aid.
PMCID: PMC3166583  PMID: 22689275
5.  The Translational Repressor T-cell Intracellular Antigen-1 (TIA-1) is a Key Modulator of Th2 and Th17 Responses Driving Pulmonary Inflammation Induced by Exposure to House Dust Mite 
Immunology letters  2012;146(1-2):8-14.
T-cell Intracellular Antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1−/−) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1−/− mice also had higher levels of Df-specific IgE and IgG1 in serum and ex vivo restimulated Tia-1−/− lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.
PMCID: PMC3407291  PMID: 22525013
T-cell Intracellular Antigen-1; allergen-mediated pulmonary inflammation; cytokines; translation
6.  A Large Cohort Study of Hypothyroidism and Hyperthyroidism in Relation to Gynecologic Cancers 
Background. Thyroid status may influence tumorigenesis of gynecologic cancers, yet epidemiologic studies of this relationship are limited and inconsistent. Methods. We evaluated the association of self-reported history of physician-diagnosed hypothyroidism and hyperthyroidism with medical-record confirmed endometrial (EC; all invasive adenocarcinomas) and ovarian cancer (OC; epithelial ovarian or peritoneal cancers) in Nurses' Health Study (NHS) from 1976 to 2010 and NHSII from 1989 to 2011. Cox proportional hazard models were used to estimate multivariable rate ratios (RRs) and 95% confidence intervals based on pooled cohort data. Results. We confirmed 1314 incident cases of EC and 1150 cases of OC. Neither a history of hypothyroidism nor hyperthyroidism was significantly associated with risk of EC or OC. However, having a history of hypothyroidism for 8+ years (median) was nonsignificantly inversely associated with EC (RR = 0.81; 95% CI = 0.63–1.04; P-trend with history duration = 0.11) and OC (RR = 0.87, 95% CI = 0.66–1.15; P-trend = 0.13). Having a history of hyperthyroidism for 6+ years (median) was non-significantly positively associated with EC (RR = 1.69; 95% CI = 0.86–3.30; P-trend = 0.12) but not OC (RR = 1.12; 95% CI = 0.46–2.72; P-trend = 0.95). Conclusions. A history of hypothyroidism or hyperthyroidism was not significantly associated with risk of EC or OC.
PMCID: PMC3728510  PMID: 23956749
7.  Epigenetic Impact of Long-Term Shiftwork: Pilot Evidence From Circadian Genes and Whole-Genome Methylation Analysis 
Chronobiology international  2011;28(10):852-861.
Epigenetic association studies have demonstrated differential promoter methylation in the core circadian genes in breast cancer cases relative to cancer-free controls. The current pilot study aims to investigate whether epigenetic changes affecting breast cancer risk could be caused by circadian disruption through exposure to light at night. Archived DNA samples extracted from whole blood of 117 female subjects from a prospective cohort conducted in Denmark were included in this study. A polymerase chain reaction (PCR)-based method was used for detection of gene-promoter methylation, whereas genome-wide methylation analysis was performed using the Illumina Infinium Methylation Chip. Long-term shiftwork resulted in the same promoter hypomethylation of CLOCK and hypermethylation of CRY2, as was previously observed in breast cancer case-control studies. Genome-wide methylation analysis further discovered widespread methylation alterations in shiftworkers, including changes in many methylation- and cancer-relevant genes. Pathway analysis of the genes with altered methylation patterns revealed several cancer-related pathways. One of the top three networks generated was designated as “DNA replication, recombination, and repair, gene expression, behavior” with ESR1 (estrogen receptor α) featured most prominently in the network, underscoring the potential breast cancer relevance of the genes differentially methylated in long-term shiftworkers. These results, although exploratory, demonstrate the first evidence of the cancer-relevant epigenetic effects of night shiftwork, which warrant further investigation. Considering there are millions of shiftworkers worldwide, understanding the effects of this exposure may lead to novel strategies for cancer prevention and new policies regulating shiftwork.
PMCID: PMC3631105  PMID: 22080730
Cancer; Circadian genes; Genome-wide methylation; Shiftwork
9.  Stronger Tests of Mechanisms Underlying Geographic Gradients of Biodiversity: Insights from the Dimensionality of Biodiversity 
PLoS ONE  2013;8(2):e56853.
Inference involving diversity gradients typically is gathered by mechanistic tests involving single dimensions of biodiversity such as species richness. Nonetheless, because traits such as geographic range size, trophic status or phenotypic characteristics are tied to a particular species, mechanistic effects driving broad diversity patterns should manifest across numerous dimensions of biodiversity. We develop an approach of stronger inference based on numerous dimensions of biodiversity and apply it to evaluate one such putative mechanism: the mid-domain effect (MDE). Species composition of 10,000-km2 grid cells was determined by overlaying geographic range maps of 133 noctilionoid bat taxa. We determined empirical diversity gradients in the Neotropics by calculating species richness and three indices each of phylogenetic, functional and phenetic diversity for each grid cell. We also created 1,000 simulated gradients of each examined metric of biodiversity based on a MDE model to estimate patterns expected if species distributions were randomly placed within the Neotropics. For each simulation run, we regressed the observed gradient onto the MDE-expected gradient. If a MDE drives empirical gradients, then coefficients of determination from such an analysis should be high, the intercept no different from zero and the slope no different than unity. Species richness gradients predicted by the MDE fit empirical patterns. The MDE produced strong spatially structured gradients of taxonomic, phylogenetic, functional and phenetic diversity. Nonetheless, expected values generated from the MDE for most dimensions of biodiversity exhibited poor fit to most empirical patterns. The MDE cannot account for most empirical patterns of biodiversity. Fuller understanding of latitudinal gradients will come from simultaneous examination of relative effects of random, environmental and historical mechanisms to better understand distribution and abundance of the current biota.
PMCID: PMC3581556  PMID: 23451099
10.  Quantifying the Effect of Metformin Treatment and Dose on Glycemic Control 
Diabetes Care  2012;35(2):446-454.
Metformin is the first-line oral medication recommended for glycemic control in patients with type 2 diabetes. We reviewed the literature to quantify the effect of metformin treatment on glycated hemoglobin (HbA1c) levels in all types of diabetes and examine the impact of differing doses on glycemic control.
MEDLINE, EMBASE, and the Cochrane Library were searched from 1950 to June 2010 for trials of at least 12 weeks’ duration in which diabetic patients were treated with either metformin monotherapy or as an add-on therapy. Data on change in HbA1c were pooled in a meta-analysis. Data from dose-comparison trials were separately pooled.
A total of 35 trials were identified for the main analysis and 7 for the dose-comparison analysis. Metformin monotherapy lowered HbA1c by 1.12% (95% CI 0.92–1.32; I2 = 80%) versus placebo, metformin added to oral therapy lowered HbA1c by 0.95% (0.77–1.13; I2 = 77%) versus placebo added to oral therapy, and metformin added to insulin therapy lowered HbA1c by 0.60% (0.30–0.91; I2 = 79.8%) versus insulin only. There was a significantly greater reduction in HbA1c using higher doses of metformin compared with lower doses of metformin with no significant increase in side effects.
Evidence supports the effectiveness of metformin therapy in a clinically important lowering of HbA1c used as monotherapy and in combination with other therapeutic agents. There is potential for using higher doses of metformin to maximize glycemic control in diabetic patients without increasing gastrointestinal effects.
PMCID: PMC3263873  PMID: 22275444
11.  How well do clinical prediction rules perform in identifying serious infections in acutely ill children across an international network of ambulatory care datasets? 
BMC Medicine  2013;11:10.
Diagnosing serious infections in children is challenging, because of the low incidence of such infections and their non-specific presentation early in the course of illness. Prediction rules are promoted as a means to improve recognition of serious infections. A recent systematic review identified seven clinical prediction rules, of which only one had been prospectively validated, calling into question their appropriateness for clinical practice. We aimed to examine the diagnostic accuracy of these rules in multiple ambulatory care populations in Europe.
Four clinical prediction rules and two national guidelines, based on signs and symptoms, were validated retrospectively in seven individual patient datasets from primary care and emergency departments, comprising 11,023 children from the UK, the Netherlands, and Belgium. The accuracy of each rule was tested, with pre-test and post-test probabilities displayed using dumbbell plots, with serious infection settings stratified as low prevalence (LP; <5%), intermediate prevalence (IP; 5 to 20%), and high prevalence (HP; >20%) . In LP and IP settings, sensitivity should be >90% for effective ruling out infection.
In LP settings, a five-stage decision tree and a pneumonia rule had sensitivities of >90% (at a negative likelihood ratio (NLR) of < 0.2) for ruling out serious infections, whereas the sensitivities of a meningitis rule and the Yale Observation Scale (YOS) varied widely, between 33 and 100%. In IP settings, the five-stage decision tree, the pneumonia rule, and YOS had sensitivities between 22 and 88%, with NLR ranging from 0.3 to 0.8. In an HP setting, the five-stage decision tree provided a sensitivity of 23%. In LP or IP settings, the sensitivities of the National Institute for Clinical Excellence guideline for feverish illness and the Dutch College of General Practitioners alarm symptoms ranged from 81 to 100%.
None of the clinical prediction rules examined in this study provided perfect diagnostic accuracy. In LP or IP settings, prediction rules and evidence-based guidelines had high sensitivity, providing promising rule-out value for serious infections in these datasets, although all had a percentage of residual uncertainty. Additional clinical assessment or testing such as point-of-care laboratory tests may be needed to increase clinical certainty. None of the prediction rules identified seemed to be valuable for HP settings such as emergency departments.
PMCID: PMC3566974  PMID: 23320738
clinical prediction rules; serious infection in children; external validation; NICE guidelines feverish illness; Yale Observation Scale; diagnostic accuracy
12.  Pulmonary Sarcoidosis following Etanercept Treatment 
Case Reports in Rheumatology  2012;2012:724013.
Tumour necrosis factor (TNF) is an important cytokine involved in the pathology of a number of inflammatory conditions, and thus blockade with anti-TNF therapies is becoming the cornerstone in managing such diseases. With increasing use, evidence is collected for the association of sarcoid-like granulomatous disease developing after the initiation of anti-TNF-α therapy, with disease reversal after discontinuation.
PMCID: PMC3540653  PMID: 23320239
13.  Exhibition review 
PMCID: PMC3223768
14.  Interleukin-33 Primes Mast Cells for Activation by IgG Immune Complexes 
PLoS ONE  2012;7(10):e47252.
Mast cells (MCs) are heterogeneous cells whose phenotype is modulated by signals received from the local microenvironment. Recent studies have identified the mesenchymal-derived cytokine IL-33 as a potent direct activator of MCs, as well as regulator of their effector phenotype, and have implicated this activity in the ability of mast cells to contribute to murine experimental arthritis. We explored the hypothesis that IL-33 enables participation of synovial MCs in murine K/BxN arthritis by promoting their activation by IgG immune complexes. Compared to wild-type (WT) control mice, transgenic animals lacking the IL-33 receptor ST2 exhibited impaired MC-dependent immune complex-induced vascular permeability (flare) and attenuated K/BxN arthritis. Whereas participation of MCs in this model is mediated by the activating IgG receptor FcγRIII, we pre-incubated bone marrow-derived MCs with IL-33 and found not only direct induction of cytokine release but also a marked increase in FcγRIII-driven production of critical arthritogenic mediators including IL-1β and CXCL2. This “priming” effect was associated with mRNA accumulation rather than altered expression of Fcγ receptors, could be mimicked by co-culture of WT but not ST2−/− MCs with synovial fibroblasts, and was blocked by antibodies against IL-33. In turn, WT but not ST2−/− MCs augmented fibroblast expression of IL-33, forming a positive feedback circuit. Together, these findings confirm a novel role for IL-33 as an amplifier of IgG immune complex-mediated inflammation and identify a potential MC-fibroblast amplification loop dependent on IL-33 and ST2.
PMCID: PMC3469528  PMID: 23071771
15.  Biomarkers of organophosphorus (OP) exposures in humans 
Neurotoxicology  2011;32(5):656-660.
There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of “biomarker proteins” that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS.
PMCID: PMC3279568  PMID: 21767566
Biomarkers; Tricresyl phosphate; CBDP; Butyrylcholinesterase; Acylpeptide hydrolase; Aerotoxic syndrome
16.  Next-generation sequencing at Merck-Boston 
BMC Proceedings  2012;6(Suppl 6):P36.
PMCID: PMC3467709
17.  Type and accuracy of sphygmomanometers in primary care: a cross-sectional observational study 
The British Journal of General Practice  2011;61(590):e598-e603.
Previous studies identified worrying levels of sphygmomanometer inaccuracy and have not been repeated in the era of digital measurement of blood pressure
To establish the type and accuracy of sphygmomanometers in current use
Design and setting
Cross-sectional, observational study in 38 Oxfordshire primary care practices
Sphygmomanometers were evaluated between 50 and 250 mmHg, using Omron PA350 or Scandmed 950831-2 pressure meters.
Six hundred and four sphygmomanometers were identified: 323 digital (53%), 192 aneroid (32%), 79 mercury (13%), and 10 hybrid (2%) devices. Of these, 584 (97%) could be fully tested. Overall, 503/584 (86%) were within 3 mmHg of the reference, 77/584 (13%) had one or more errorof 4-9 mmHg, and 4/584 (<1%) had one or more errorof more than 10 mmHg. Mercury (71/75, 95%) and digital (272/308, 88%) devices were more likely to be within 3 mmHg of the reference standard than aneroid models (150/191, 78%) (Fisher's exact test P = 0.001). Donated aneroid devices from the pharmaceutical industry performed significantly worse: 10/23 (43%) within 3 mmHg of standard compared to 140/168 (83%) aneroid models from recognised manufacturers (Fisher's exact test P<0.001). No significant difference was found in performance between manufacturers within each device type, for either aneroid (Fisher's exact test P = 0.96) or digital (Fisher's exact test P = 0.7) devices.
Digital sphygmomanometers have largely replaced mercury models in primary care and have equivalent accuracy. Aneroid devices have higherfailure rates than other device types; this appears to be largely accounted forby models from indiscernible manufacturers. Given the availability of inexpensive and accurate digital models, GPs could consider replacing aneroid devices with digital equivalents, especially for home visiting.
PMCID: PMC3162183  PMID: 22152749
blood pressure determination; hypertension; general practice; quality assurance, health care
18.  Relative effects of time for speciation and tropical niche conservatism on the latitudinal diversity gradient of phyllostomid bats 
Determinants of contemporary patterns of diversity, particularly those spanning extensive latitudinal gradients, are some of the most intensely debated issues in ecology. Recently, focus has shifted from a contemporary environmental perspective to a historical one in an attempt to better understand the construction of latitudinal gradients. Although the vast majority of research on historical mechanisms has focused on tropical niche conservatism (TNC), other historical scenarios could produce similar latitudinal gradients. Herein, I formalize predictions to distinguish between two such historical processes—namely time for speciation (TFS) and TNC—and test relative support based on diversity gradients of New World bats. TFS and TNC are distinctly spatial and environmental mechanisms, respectively. Nonetheless, because of the way that environmental characteristics vary spatially, these two mechanisms are hard to distinguish. Evidence provided herein suggests that TNC has had a more important effect than TFS in determining diversity gradients of New World bats. Indeed, relative effects of different historical mechanisms, as well as relative effects of historical and contemporary environmental determinants, are probably context-dependent. Future research should move away from attempting to identify the mechanism with primacy and instead attempt to understand the particular contexts in which different mechanisms have greater influence on diversity gradients.
PMCID: PMC3125621  PMID: 21208951
bats; biodiversity; historical process; latitudinal diversity gradient; time for speciation; tropical niche conservatism
19.  Mast Cell Tryptase Deficiency Attenuates Mouse Abdominal Aortic Aneurysm Formation 
Circulation research  2011;108(11):1316-1327.
Mast cells (MCs) contribute to formation of abdominal aortic aneurysms (AAAs) by producing biologically active mediators. Tryptase is the most abundant MC granule protein and participates in MC activation, protease maturation, leukocyte recruitment, and angiogenesis — all processes critical to AAA pathogenesis.
To test the hypothesis that tryptase functions directly in AAA formation.
Methods and Results
Immunoreactive tryptase localized in the media and adventitia of human and mouse AAA lesions. Serum tryptase levels correlated significantly with the annual expansion rate of AAA before (r=0.30, P=0.003) and after (r=0.29, P=0.005) adjustment for common AAA risk factors in a patient follow-up study, and associated with risks for later surgical repair or overall mortality before (P=0.009, P=0.065) and after (P=0.004, P=0.001) the adjustment. Using MC protease-6–deficient mice (Mcpt6−/−) and experimental AAAs induced by aortic elastase perfusion, we proved a direct role of this tryptase in AAA pathogenesis. While all wild-type (WT) mice developed AAA at 14 or 56 days post-perfusion, Mcpt6−/− mice had full protection. AAA lesions from Mcpt6−/− mice contained fewer inflammatory and apoptotic cells, and lower chemokine levels than those from WT mice. MC from WT mice restored reduced AAA lesions and lesion inflammatory cell content in MC–deficient KitW-sh/W-sh mice, but those prepared from Mcpt6−/− mice did not. Mechanistic studies demonstrated that tryptase deficiency affected endothelial cell (EC) chemokine and cytokine expression, monocyte transmigration, smooth-muscle cell apoptosis, and MC and AAA lesion cysteinyl cathepsin expression and activities.
This study establishes the direct participation of MC tryptase in the pathogenesis of experimental AAAs, and suggests that levels of this protease can serve as a novel biomarker for abdominal aortic expansion.
PMCID: PMC3108141  PMID: 21493897
abdominal aortic aneurysm; tryptase; mMCP-6; macrophage; T cell; apoptosis
20.  Survey Sequencing Reveals Elevated DNA Transposon Activity, Novel Elements, and Variation in Repetitive Landscapes among Vesper Bats 
Genome Biology and Evolution  2012;4(4):575-585.
The repetitive landscapes of mammalian genomes typically display high Class I (retrotransposon) transposable element (TE) content, which usually comprises around half of the genome. In contrast, the Class II (DNA transposon) contribution is typically small (<3% in model mammals). Most mammalian genomes exhibit a precipitous decline in Class II activity beginning roughly 40 Ma. The first signs of more recently active mammalian Class II TEs were obtained from the little brown bat, Myotis lucifugus, and are reflected by higher genome content (∼5%). To aid in determining taxonomic limits and potential impacts of this elevated Class II activity, we performed 454 survey sequencing of a second Myotis species as well as four additional taxa within the family Vespertilionidae and an outgroup species from Phyllostomidae. Graph-based clustering methods were used to reconstruct the major repeat families present in each species and novel elements were identified in several taxa. Retrotransposons remained the dominant group with regard to overall genome mass. Elevated Class II TE composition (3–4%) was observed in all five vesper bats, while less than 0.5% of the phyllostomid reads were identified as Class II derived. Differences in satellite DNA and Class I TE content are also described among vespertilionid taxa. These analyses present the first cohesive description of TE evolution across closely related mammalian species, revealing genome-scale differences in TE content within a single family.
PMCID: PMC3342881  PMID: 22491057
transposon; survey sequencing; Chiroptera
21.  The inflammatory response following an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5 
A second degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to non-muscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-KitW/KitWv mice which lack MCs in a context of other defects due to a mutation of the KIT receptor. To further address a MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury following a second degree scald burn while mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or the MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficent mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first h post burn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing non-redundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.
PMCID: PMC3058366  PMID: 21076070
22.  CLOCK in breast tumorigenesis: evidence from genetic, epigenetic, and transcriptional profiling analyses 
Cancer research  2010;70(4):1459-1468.
As transcriptional regulators, the genes responsible for maintaining circadian rhythm exert influence in a variety of biological processes. Recently, it has been suggested that the core circadian genes may play a role in breast tumorigenesis, possibly by influencing hormone regulation or other cancer-relevant pathways. Here, we examine the role of the central circadian regulator CLOCK in breast cancer by conducting a genetic and epigenetic association study, as well as transcriptional profiling arrays and a pathway-based network analysis. Significant associations were detected between CLOCK tagging SNPs and breast cancer risk, with apparent effect modification by ER/PR status. Furthermore, hypermethylation in the CLOCK promoter was found to reduce breast cancer risk, and these findings were corroborated by publicly available tissue array data, which showed lower levels of CLOCK expression in healthy controls relative to normal or tumor tissue from breast cancer patients. Finally, we silenced CLOCK in vitro and performed a whole genome expression microarray and pathway analysis, which identified a cancer-relevant network of transcripts with altered expression following CLOCK gene knockdown. These findings support the hypothesis that circadian genes may be relevant for tumorigenesis, and suggest that circadian gene variants may represent a novel panel of breast cancer susceptibility biomarkers.
PMCID: PMC3188957  PMID: 20124474
CLOCK; Circadian Genetics; Breast Cancer; Genetic variants; Epigenetic variants
23.  The core circadian gene cryptochrome 2 influences breast cancer risk, possibly by mediating hormone signaling 
As transcriptional regulators, circadian genes have the potential to influence a variety of biological pathways, including many cancer-related processes. Cryptochrome 2 (CRY2) is essential for proper circadian timing, and is a key component of the circadian regulatory feedback loop. Here, we report findings from genetic, epigenetic, loss-of-function, and transcriptional profiling analyses of CRY2 in breast cancer. Six SNPs in CRY2 were identified for genotyping in a case-control population (N=441 cases and N=479 controls), and three SNPs (rs11038689, rs7123390, and rs1401417) were significantly associated with postmenopausal breast cancer risk, with significant effect modification by menopausal status (dominant model for rs11038689: odds ratio (OR) = 0.71, 95% confidence interval (CI): 0.51–0.99, P for trend = 0.028; homozygous variants for rs7123390: OR = 0.44, 95% CI: 0.22–0.86, P for trend = 0.028; and rs1401417, OR=0.44, 95% CI: 0.21–0.92, P for trend = 0.017). Interestingly, this association was only evident in women with estrogen and progesterone receptor (ER/PR) negative breast tumors, but not with ER/PR positive tumors. Breast cancer patients also had significantly higher levels of CRY2 promoter methylation relative to controls, which is consistent with tissue array data showing lower levels of CRY2 expression in tumor tissue relative to adjacent normal tissue. Furthermore, in vitro analyses identified a number of breast cancer-relevant genes which displayed altered expression following CRY2 knockdown. These findings suggest a role for CRY2 in breast tumorigenesis, and provide further evidence that the circadian system may be an important modulator of hormone-related cancer susceptibility.
PMCID: PMC3175631  PMID: 20233903
CRY2; Breast Cancer; Circadian Biomarker; Methylation; Microarray
24.  Clock-cancer connection in Non-Hodgkin’s Lymphoma: a genetic association study and pathway analysis of the circadian gene Cryptochrome 2 
Cancer research  2009;69(8):3605-3613.
Circadian genes have the potential to influence a variety of cancer-related biological pathways, including immune regulation, which may influence susceptibility to non-Hodgkin’s lymphoma (NHL). However, few studies have examined the role of circadian genes in lymphomagenesis. The current study examined Cryptochrome 2 (CRY2), a core circadian gene and transcriptional repressor, as a potential circadian biomarker for NHL. We first performed genetic association analyses of tagging SNPs in CRY2 and NHL risk using DNA samples from a population-based case-control study (N= 455 cases and 527 controls). Three SNPs were found to be significantly associated with risk of NHL when combining all subtypes (dbSNP IDs, odds ratios (ORs), and 95% confidence intervals: rs11038689, OR=2.34 (1.28-4.27), P=0.006; rs7123390, OR=2.40 (1.39-4.13), P=0.002; and rs1401417, OR=2.97 (1.57-5.63), P=0.001). Each of these associations remained significant when restricting the analysis to B-Cell cases and when further restricting to follicular lymphomas. An analysis of CRY2 diplotypes confirmed these significant findings. To further determine the functional impact of CRY2, we silenced the gene in vitro and performed a whole genome expression microarray. A pathway-based analysis showed that genes significantly altered by CRY2 knockdown formed networks associated with immune response and hematological system development. In addition, these genes were predicted to have significant impacts on several disease processes, including cancer (B-H P-value=3.75E-9) and hematological disease (B-H P=8.01E-8). In conclusion, both genetic association and functional analyses suggest that the circadian gene CRY2 may play an important role in NHL development.
PMCID: PMC3175639  PMID: 19318546
CRY2; NHL; Circadian Genetics

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