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1.  Trends in serum creatinine testing in Oxfordshire, UK, 1993–2013: a population-based cohort study 
BMJ Open  2015;5(12):e009459.
Objectives
To determine how many kidney function tests are done, on whom, how frequently they are performed and how they have changed over time.
Design
Retrospective study of all serum creatinine, urine albumin and urine creatinine tests.
Setting
Primary and secondary care in Oxfordshire from 1993 to 2013.
Participants
Unselected population of 1 220 447 people.
Main outcome measures
The total number of creatinine and urinary protein tests ordered from primary and secondary care and the number of tests per year stratified by categories of estimated glomerular filtration rate (eGFR). The frequency of testing in patients having their kidney function monitored.
Results
Creatinine requests from primary care increased steadily from 1997 and exceeded 220 000 requests in 2013. Tests corresponding to normal kidney function (eGFR >60/mL/min/1.73 m2) constituted 59% of all kidney function tests in 1993 and accounted for 83% of all tests in 2013. Test corresponding to chronic kidney disease (CKD) stages 3–5 declined after 2007. Reduced kidney function, albuminuria, male gender, diabetes and age were independently associated with more frequent monitoring. For a female patient between 61 and 80 years and with stage 3a CKD, the average number of serum creatinine tests (95% CI) was 3.23/year (3.19 to 3.26) and for a similar woman with diabetes, the average number of tests was 5.50 (5.44 to 5.56) tests per year.
Conclusions
There has been a large increase in the number of kidney function tests over the past two decades. However, we found little evidence that this increase is detecting more CKD. Tests are becoming more frequent in people with and without evidence of renal impairment. Future work using a richer data source could help unravel the underlying reasons for the increased testing and determine how much is necessary and useful.
doi:10.1136/bmjopen-2015-009459
PMCID: PMC4691790  PMID: 26674500
PRIMARY CARE; EPIDEMIOLOGY; Chronic kidney disease; serum creatinine
2.  Ancient origin of mast cells 
The sentinel roles of mammalian mast cells (MCs) in varied infections raised the question of their evolutionary origin. We discovered that the test cells in the sea squirt Ciona intestinalis morphologically and histochemically resembled cutaneous human MCs. Like the latter, C. intestinalis test cells stored histamine and varied heparin•serine protease complexes in their granules. Moreover, they exocytosed these preformed mediators when exposed to compound 48/80. In support of the histamine data, a C. intestinalis-derived cDNA was isolated that resembled that which encodes histidine decarboxylase in human MCs. Like heparin-expressing mammalian MCs, activated test cells produced prostaglandin D2 and contained cDNAs that encode a protein that resembles the synthase needed for its biosynthesis in human MCs. The accumulated morphological, histochemical, biochemical, and molecular biology data suggest that the test cells in C. intestinalis are the counterparts of mammalian MCs that reside in varied connective tissues. The accumulated data point to an ancient origin of MCs that predates the emergence of the chordates >500 million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity.
doi:10.1016/j.bbrc.2014.07.124
PMCID: PMC4145527  PMID: 25094046
mast cell; Ciona intestinalis; heparin; serine protease; histamine; prostaglandin D2
4.  Prevalence and Prognostic Role of Triple-Negative Breast Cancer by Race: A Surveillance Study 
Clinical breast cancer  2011;11(5):332-341.
A possible explanation for the relatively poor survival from breast cancer among blacks is the much higher rate of the adverse Triple-Negative sub-type. In a study of 1372 patients, blacks had twice the risk of death compared to whites among those with advanced cancer whether or not tumors were Triple-Negative. More research is still warranted to determine why blacks with advanced, but not local, breast cancer have a consistently higher rate of death.
Introduction
Emerging research suggests a substantially greater prevalence of the adverse triple-negative (TN) subtype (human epidermal growth factor receptor [HER]2−, estrogen receptor [ER]−, and progesterone receptor [PR])−) among black patients with breast cancer. No reports however have been generated from a statewide cancer registry.
Patients and Methods
The study consisted of all black patients (N = 643) and a random sample of white patients (n = 719) diagnosed with primary invasive breast cancer (2000–2003) listed in the National Cancer Institute–Surveillance Epidemiology and End Results (NCI-SEER) Connecticut Tumor Registry (CTR). HER2 status was obtained from pathology reports submitted to the registry. Remaining data were obtained from the registry database.
Results
TN tumors were more prevalent in black compared with white patients (30.8% vs. 11.2%, respectively; P < .001.) There was a 2-fold greater frequency of ER− and PR− phenotypes among black patients, but HER2 status did not differ by race. Patients with lobular cancer were less likely to have TN breast cancer compared with patients with ductal tumors (odds ratio [OR] = 0.23; 95% confidence interval [CI], 0.10–0.58). Among patients with regional disease, black patients exhibited increased risk of death (relative risk [RR] = 2.71; 95% CI, 1.48–4.97) independent of TN status. No survival disparity was found among patients with local disease.
Discussion
These registry-based data corroborate reports that TN breast cancer varies substantially by race and histologic subtype. A survival disparity among patients with advanced disease, but not local disease, casts some doubt on TN status as an explanation for differences.
Conclusion
More research is warranted to understand why black patients with advanced breast cancer may be at increased risk for death whether or not their tumors express the TN phenotype.
doi:10.1016/j.clbc.2011.04.004
PMCID: PMC4459583  PMID: 21729670
Biologic markers; Breast neoplasms; Health status disparities; HER2; Race/ethnicity; Triple negative breast cancer
5.  Targeted Capture of Phylogenetically Informative Ves SINE Insertions in Genus Myotis 
Genome Biology and Evolution  2015;7(6):1664-1675.
Identification of retrotransposon insertions in nonmodel taxa can be technically challenging and costly. This has inhibited progress in understanding retrotransposon insertion dynamics outside of a few well-studied species. To address this problem, we have extended a retrotransposon-based capture and sequence method (ME-Scan [mobile element scanning]) to identify insertions belonging to the Ves family of short interspersed elements (SINEs) across seven species of the bat genus Myotis. We identified between 120,000 and 143,000 SINE insertions in six taxa lacking a draft genome by comparing to the M. lucifugus reference genome. On average, each Ves insertion was sequenced to 129.6 × coverage. When mapped back to the M. lucifugus reference genome, all insertions were confidently assigned within a 10-bp window. Polymorphic Ves insertions were identified in each taxon based on their mapped locations. Using cross-species comparisons and the identified insertion positions, a presence–absence matrix was created for approximately 796,000 insertions. Dollo parsimony analysis of more than 85,000 phylogenetically informative insertions recovered strongly supported, monophyletic clades that correspond with the biogeography of each taxa. This phylogeny is similar to previously published mitochondrial phylogenies, with the exception of the placement of M. vivesi. These results support the utility of our variation on ME-Scan to identify polymorphic retrotransposon insertions in taxa without a reference genome and for large-scale retrotransposon-based phylogenetics.
doi:10.1093/gbe/evv099
PMCID: PMC4494050  PMID: 26014613
rare genomic events; Dollo parsimony; retrotransposon; phylogenetics; Myotis lucifugus
6.  Heterogeneity of Prognostic Studies of 24-Hour Blood Pressure Variability: Systematic Review and Meta-Analysis 
PLoS ONE  2015;10(5):e0126375.
In addition to mean blood pressure, blood pressure variability is hypothesized to have important prognostic value in evaluating cardiovascular risk. We aimed to assess the prognostic value of blood pressure variability within 24 hours. Using MEDLINE, EMBASE and Cochrane Library to April 2013, we conducted a systematic review of prospective studies of adults, with at least one year follow-up and any day, night or 24-hour blood pressure variability measure as a predictor of one or more of the following outcomes: all-cause mortality, cardiovascular mortality, all cardiovascular events, stroke and coronary heart disease. We examined how blood pressure variability is defined and how its prognostic use is reported. We analysed relative risks adjusted for covariates including the appropriate mean blood pressure and considered the potential for meta-analysis. Our analysis of methods included 24 studies and analysis of predictions included 16 studies. There were 36 different measures of blood pressure variability and 13 definitions of night- and day-time periods. Median follow-up was 5.5 years (interquartile range 4.2–7.0). Comparing measures of dispersion, coefficient of variation was less well researched than standard deviation. Night dipping based on percentage change was the most researched measure and the only measure for which data could be meaningfully pooled. Night dipping or lower night-time blood pressure was associated with lower risk of cardiovascular events. The interpretation and use in clinical practice of 24-hour blood pressure variability, as an important prognostic indicator of cardiovascular events, is hampered by insufficient evidence and divergent methodologies. We recommend greater standardisation of methods.
doi:10.1371/journal.pone.0126375
PMCID: PMC4435972  PMID: 25984791
7.  Differential SINE evolution in vesper and non-vesper bats 
Mobile DNA  2015;6:10.
Background
Short interspersed elements (SINEs) have a powerful influence on genome evolution and can be useful markers for phylogenetic inference and population genetic analyses. In this study, we examined survey sequence and whole genome data to determine the evolutionary dynamics of Ves SINEs in the genomes of 11 bats, nine from Vespertilionidae.
Results
We identified 41 subfamilies of Ves and linked several to specific lineages. We also revealed substantial differences among lineages including the observation that Ves accumulation and Ves subfamily diversity is significantly higher in vesper as opposed to non-vesper bats. This is especially interesting when one considers the increased transposable element diversity of vesper bats in general.
Conclusions
Our data suggest that survey sequencing and genome mining are valuable tools to investigate SINE evolution among related lineages and can provide substantial information about the ability of SINEs to proliferate in diverse genomes. This method would also be a useful first step in determining which subfamilies would be the best to target when developing SINEs as markers for phylogenetic and population genetic analyses.
Electronic supplementary material
The online version of this article (doi:10.1186/s13100-015-0038-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13100-015-0038-4
PMCID: PMC4436864  PMID: 25991928
8.  The Multifaceted Mast Cell in Inflammatory Bowel Disease 
Inflammatory bowel diseases  2014;20(12):2364-2378.
Mast cells (MCs) are tissue-resident immune cells that carry out protective roles against pathogens. In disease states, such as inflammatory bowel disease, these granulocytes release a diverse array of mediators that contribute to inflammatory processes. They also participate in wound repair and tissue remodeling. In this review, the composition of MCs and how their phenotypes can be altered during inflammation of the gastrointestinal tract is detailed. Animal and human clinical studies that have implicated the participation of MCs in inflammatory bowel disease are reviewed, including the contribution of the cell’s mediators to clinical symptoms, stress-triggered inflammation, and fistula and strictures. Studies that have focused on negating the proinflammatory roles of MCs and their mediators in animal models suggest new targets for therapies for patients with inflammatory bowel disease.
doi:10.1097/MIB.0000000000000142
PMCID: PMC4428674  PMID: 25401721
inflammatory bowel disease; ulcerative colitis; Crohn’s disease; mast cell; tryptase; chymase
9.  Breast Cancer and Circadian Disruption from Electric Lighting in the Modern World 
Breast cancer is the leading cause of cancer death among women worldwide and there is only limited explanation of why. Risk is highest in the most industrialized countries but also rising rapidly in the developing world. Known risk factors account for only a portion of the incidence in the high risk populations, and there has been considerable speculation, and many false leads, on other possibly major determinants of risk such as dietary fat. A hallmark of industrialization is the increasing use of electricity to light the night, both within the home and without. It has only recently become clear that this evolutionarily new, and thereby unnatural exposure can disrupt human circadian rhythmicity, of which three salient features are melatonin production, sleep, and the circadian clock. A convergence of research in cells, rodents, and humans suggests that the health consequences of circadian disruption may be substantial. An innovative experimental model has shown that light at night markedly increases growth of human breast cancer xenografts in rats. In humans, the theory that light exposure at night increases breast cancer risk leads to specific predictions that are being tested epidemiologically: evidence has accumulated on risk in shift workers, risk in blind women, and impact of sleep duration on risk. If electric light at night does explain a portion of the breast cancer burden then there are practical interventions that can be implemented, including more selective use of light, and adoption of recent advances in lighting technology and application.
doi:10.3322/caac.21218
PMCID: PMC4038658  PMID: 24604162
10.  Clinical prediction rules in practice: review of clinical guidelines and survey of GPs 
The British Journal of General Practice  2014;64(621):e233-e242.
Background
The publication of clinical prediction rules (CPRs) studies has risen significantly. It is unclear if this reflects increasing usage of these tools in clinical practice or how this may vary across clinical areas.
Aim
To review clinical guidelines in selected areas and survey GPs in order to explore CPR usefulness in the opinion of experts and use at the point of care.
Design and setting
A review of clinical guidelines and survey of UK GPs.
Method
Clinical guidelines in eight clinical domains with published CPRs were reviewed for recommendations to use CPRs including primary prevention of cardiovascular disease, transient ischaemic attack (TIA) and stroke, diabetes mellitus, fracture risk assessment in osteoporosis, lower limb fractures, breast cancer, depression, and acute infections in childhood. An online survey of 401 UK GPs was also conducted.
Results
Guideline review: Of 7637 records screened by title and/or abstract, 243 clinical guidelines met inclusion criteria. CPRs were most commonly recommended in guidelines regarding primary prevention of cardiovascular disease (67%) and depression (67%). There was little consensus across various clinical guidelines as to which CPR to use preferentially. Survey: Of 401 responders to the GP survey, most were aware of and applied named CPRs in the clinical areas of cardiovascular disease and depression. The commonest reasons for using CPRs were to guide management and conform to local policy requirements.
Conclusion
GPs use CPRs to guide management but also to comply with local policy requirements. Future research could focus on which clinical areas clinicians would most benefit from CPRs and promoting the use of robust, externally validated CPRs.
doi:10.3399/bjgp14X677860
PMCID: PMC3964449  PMID: 24686888
clinical prediction rules; clinical guidelines; survey
11.  HCV Genotyping from NGS Short Reads and Its Application in Genotype Detection from HCV Mixed Infected Plasma 
PLoS ONE  2015;10(4):e0122082.
Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection.
doi:10.1371/journal.pone.0122082
PMCID: PMC4382110  PMID: 25830316
12.  Mouse Mast Cell Protease 4 and 5 Mediate Epidermal Injury Through Disruption of Tight Junctions 
We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on non-redundant functions of mouse MC protease 4 (mMCP4) and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, over the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury while mMCP4−/− mice showed no significant changes. Taken together, these findings reveal that a second degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the non-redundant presence of mMCP4 and mMCP5.
doi:10.4049/jimmunol.1301794
PMCID: PMC3970423  PMID: 24523504
13.  The science of clinical practice: disease diagnosis or patient prognosis? Evidence about “what is likely to happen” should shape clinical practice 
BMC Medicine  2015;13:20.
Background
Diagnosis is the traditional basis for decision-making in clinical practice. Evidence is often lacking about future benefits and harms of these decisions for patients diagnosed with and without disease. We propose that a model of clinical practice focused on patient prognosis and predicting the likelihood of future outcomes may be more useful.
Discussion
Disease diagnosis can provide crucial information for clinical decisions that influence outcome in serious acute illness. However, the central role of diagnosis in clinical practice is challenged by evidence that it does not always benefit patients and that factors other than disease are important in determining patient outcome. The concept of disease as a dichotomous ‘yes’ or ‘no’ is challenged by the frequent use of diagnostic indicators with continuous distributions, such as blood sugar, which are better understood as contributing information about the probability of a patient’s future outcome. Moreover, many illnesses, such as chronic fatigue, cannot usefully be labelled from a disease-diagnosis perspective. In such cases, a prognostic model provides an alternative framework for clinical practice that extends beyond disease and diagnosis and incorporates a wide range of information to predict future patient outcomes and to guide decisions to improve them. Such information embraces non-disease factors and genetic and other biomarkers which influence outcome.
Summary
Patient prognosis can provide the framework for modern clinical practice to integrate information from the expanding biological, social, and clinical database for more effective and efficient care.
doi:10.1186/s12916-014-0265-4
PMCID: PMC4311412  PMID: 25637245
Clinical decision-making; Contested diagnoses; Diagnosis; Evidence-based medicine; Information; Outcomes of care; Overdiagnosis; Prognosis; Stratified medicine
14.  Diagnostic accuracy study of three alcohol breathalysers marketed for sale to the public 
BMJ Open  2014;4(12):e005811.
Objectives
To assess the diagnostic accuracy of three personal breathalyser devices available for sale to the public marketed to test safety to drive after drinking alcohol.
Design
Prospective comparative diagnostic accuracy study comparing two single-use breathalysers and one digital multiuse breathalyser (index tests) to a police breathalyser (reference test).
Setting
Establishments licensed to serve alcohol in a UK city.
Participants
Of 222 participants recruited, 208 were included in the main analysis. Participants were eligible if they were 18 years old or over, had consumed alcohol and were not intending to drive within the following 6 h.
Outcome measures
Sensitivity and specificity of the breathalysers for the detection of being at or over the UK legal driving limit (35 µg/100 mL breath alcohol concentration).
Results
18% of participants (38/208) were at or over the UK driving limit according to the police breathalyser. The digital multiuse breathalyser had a sensitivity of 89.5% (95% CI 75.9% to 95.8%) and a specificity of 64.1% (95% CI 56.6% to 71.0%). The single-use breathalysers had a sensitivity of 94.7% (95% CI 75.4% to 99.1%) and 26.3% (95% CI 11.8% to 48.8%), and a specificity of 50.6% (95% CI 40.4% to 60.7%) and 97.5% (95% CI 91.4% to 99.3%), respectively. Self-reported alcohol consumption threshold of 5 UK units or fewer had a higher sensitivity than all personal breathalysers.
Conclusions
One alcohol breathalyser had sensitivity of 26%, corresponding to false reassurance for approximately one person in four who is over the limit by the reference standard, at least on the evening of drinking alcohol. The other devices tested had 90% sensitivity or higher. All estimates were subject to uncertainty. There is no clearly defined minimum sensitivity for this safety-critical application. We conclude that current regulatory frameworks do not ensure high sensitivity for these devices marketed to consumers for a decision with potentially catastrophic consequences.
doi:10.1136/bmjopen-2014-005811
PMCID: PMC4281544  PMID: 25526794
15.  Shiftwork and Prostate-Specific Antigen in the National Health and Nutrition Examination Survey 
Background
Shiftwork has been implicated as a risk factor for prostate cancer. Results from prior studies have been mixed but generally support an association between circadian disruption and prostate cancer. Our aim was to investigate the relationship between shiftwork and prostate-specific antigen (PSA) test obtained as part of the National Health and Nutrition Examination Survey (NHANES) study.
Methods
We combined three NHANES surveys (2005–2010) to obtain current work schedule among employed men aged 40 to 65 years with no prior history of cancer (except nonmelanoma skin cancer). Men who reported working regular night shifts or rotating shifts were considered shiftworkers. We obtained the total and percentage free PSA test results for these men and dichotomized total PSA into less than 4.0ng/mL or 4.0ng/mL or greater and total PSA of 4.0ng/mL or greater combined with percentage free PSA less than or equal to 25%. Using multivariable logistic regression models, we compared PSA level among current shiftworkers and nonshiftworkers. All statistical tests were two-sided.
Results
We found a statistically significant, age-adjusted association between current shiftwork and elevated PSA at the 4.0ng/mL or greater level (odds ratio = 2.48, 95% confidence interval [CI] = 1.08 to 5.70; P = .03). The confounder-adjusted odds ratio was 2.62 (95% CI = 1.16 to 5.95; P = .02). The confounder-adjusted odds ratio for those with total PSA of 4.0ng/mL or greater and free PSA less than or equal to 25% was 3.13 (95% CI = 1.38 to 7.09; P = .01).
Conclusions
We observed a strong positive association with shiftwork and elevated PSA level. Our data support the notion that sleep or circadian disruption is associated with elevated PSA, indicating that shiftworking men likely have an increased risk of developing prostate cancer.
doi:10.1093/jnci/djt169
PMCID: PMC3859215  PMID: 23943864
16.  All-Cause and Cardiovascular Mortality in Middle-Aged People With Type 2 Diabetes Compared With People Without Diabetes in a Large U.K. Primary Care Database 
Diabetes Care  2013;36(8):2366-2371.
OBJECTIVE
Middle-aged people with diabetes have been reported to have significantly higher risks of cardiovascular events than people without diabetes. However, recent falls in cardiovascular disease rates and more active management of risk factors may have abolished the increased risk. We aimed to provide an up-to-date assessment of the relative risks associated with type 2 diabetes of all-cause and cardiovascular mortality in middle-aged people in the U.K.
RESEARCH DESIGN AND METHODS
Using data from the General Practice Research Database, from 2004 to 2010, we conducted a cohort study of 87,098 people, 40–65 years of age at baseline, comparing 21,798 with type 2 diabetes and 65,300 without diabetes, matched on age, sex, and general practice. We produced hazard ratios (HRs) for mortality and compared rates of blood pressure testing, cholesterol monitoring, and use of aspirin, statins, and antihypertensive drugs.
RESULTS
People with type 2 diabetes, compared with people without diabetes, had a twofold increased risk of all-cause mortality (HR 2.07 [95% CI 1.95–2.20], adjusted for smoking) and a threefold increased risk of cardiovascular mortality (3.25 [2.87–3.68], adjusted for smoking). Women had a higher relative risk than men, and people <55 years of age had a higher relative risk than those >55 years of age. Monitoring and medication rates were higher in those with diabetes (all P < 0.001).
CONCLUSIONS
Despite efforts to manage risk factors, administer effective treatments, and develop new therapies, middle-aged people with type 2 diabetes remain at significantly increased risk of death.
doi:10.2337/dc12-1513
PMCID: PMC3714501  PMID: 23435157
17.  Mast Cell-restricted, Tetramer-forming Tryptases Induce Aggrecanolysis in Articular Cartilage by Activating Matrix Metalloproteinase-3 and -13 Zymogens1 
Mouse mast cell protease (mMCP)-6-null C57BL/6 mice lost less aggrecan proteoglycan from the extracellular matrix of their articular cartilage during inflammatory arthritis than wild-type (WT) C57BL/6 mice, suggesting that this mast cell (MC)-specific mouse tryptase plays prominent roles in articular cartilage catabolism. We used ex vivo mouse femoral head explants to determine how mMCP-6 and its human ortholog hTryptase-β mediate aggrecanolysis. Exposure of the explants to recombinant hTryptase-β, recombinant mMCP-6, or lysates harvested from WT mouse peritoneal MCs (PMCs) significantly increased the levels of enzymatically active matrix metalloproteinases (MMP) in cartilage and significantly induced aggrecan loss into the conditioned media, relative to replicate explants exposed to medium alone or lysates collected from mMCP-6-null PMCs. Treatment of cartilage explants with tetramer-forming tryptases generated aggrecan fragments that contained C-terminal DIPEN and N-terminal FFGVG neoepitopes, consistent with MMP-dependent aggrecanolysis. In support of these data, hTryptase-β was unable to induce aggrecan release from the femoral head explants obtained from Chloe mice that resist MMP cleavage at the DIPEN↓FFGVG site in the interglobular domain of aggrecan. In addition, the abilities of mMCP-6-containing lysates from WT PMCs to induce aggrecanolysis were prevented by inhibitors of MMP-3 and MMP-13. Finally, recombinant hTryptase-β was able to activate latent pro-MMP-3 and pro-MMP-13 in vitro. The accumulated data suggest that human and mouse tetramer-forming tryptases are MMP convertases that mediate cartilage damage and the proteolytic loss of aggrecan proteoglycans in arthritis, in part, by activating the zymogen forms of MMP-3 and MMP-13 which are constitutively present in articular cartilage.
doi:10.4049/jimmunol.1300856
PMCID: PMC3727140  PMID: 23797671
mast cells; inflammation; arthritis; cartilage; aggrecan proteoglycan; matrix metalloproteinases; tryptase
18.  Development of Mast Cells and Importance of Their Tryptase and Chymase Serine Proteases in Inflammation and Wound Healing 
Advances in immunology  2014;122:211-252.
Mast cells (MCs) are active participants in blood coagulation and innate and acquired immunity. This review focuses on the development of mouse and human MCs, as well as the involvement of their granule serine proteases in inflammation and the connective tissue remodeling that occurs during the different phases of the healing process of wounded skin and other organs. The accumulated data suggest that MCs, their tryptases, and their chymases play important roles in tissue repair. While MCs initially promote healing, they can be detrimental if they are chronically stimulated or if too many MCs become activated at the same time. The possibility that MCs and their granule serine proteases contribute to the formation of keloid and hypertrophic scars makes them potential targets for therapeutic intervention in the repair of damaged skin.
doi:10.1016/B978-0-12-800267-4.00006-7
PMCID: PMC4115062  PMID: 24507159
19.  A short-term model of COPD identifies a role for mast cell tryptase 
Background
Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short time-frame.
Objectives
To create an early onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. To use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in COPD.
Methods
Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathological features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated using depletion and in vitro studies and MC protease-6 deficient mice.
Results
After just 8 weeks of smoke exposure, wild-type mice developed chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid-resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart, and increased susceptibility to respiratory infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced pro-inflammatory responses from cultured macrophages.
Conclusion
A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.
doi:10.1016/j.jaci.2012.11.053
PMCID: PMC4060894  PMID: 23380220
cigarette smoke; COPD; inflammation; emphysema; airway remodeling; lung function; macrophage; mast cell; protease; mMCP-6; hTryptase-β
20.  Epidemiological associations between iron and cardiovascular disease and diabetes 
Disruptions in iron homeostasis are linked to a broad spectrum of chronic conditions including cardiovascular, malignant, metabolic, and neurodegenerative disease. Evidence supporting this contention derives from a variety of analytical approaches, ranging from molecular to population-based studies. This review focuses on key epidemiological studies that assess the relationship between body iron status and chronic diseases, with particular emphasis on atherosclerosis ,metabolic syndrome and diabetes. Multiple surrogates have been used to measure body iron status, including serum ferritin, transferrin saturation, serum iron, and dietary iron intake. The lack of a uniform and standardized means of assessing body iron status has limited the precision of epidemiological associations. Intervention studies using depletion of iron to alter risk have been conducted. Genetic and molecular techniques have helped to explicate the biochemistry of iron metabolism at the molecular level. Plausible explanations for how iron contributes to the pathogenesis of these chronic diseases are beginning to be elucidated. Most evidence supports the hypothesis that excess iron contributes to chronic disease by fostering excess production of free radicals. Overall, epidemiological studies, reinforced by basic science experiments, provide a strong line of evidence supporting the association between iron and elevated risk of cardiovascular disease and diabetes. In this narrative review we attempt to condense the information from existing literature on this topic.
doi:10.3389/fphar.2014.00117
PMCID: PMC4033158  PMID: 24904420
iron; cardiovascular disease; diabetes mellitus; metabolic syndrome; epidemiologic studies
21.  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.
Background
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.
Methods
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.
Results
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.
Conclusions/interpretation
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.
doi:10.1371/journal.pone.0092458
PMCID: PMC3965408  PMID: 24667212
22.  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
23.  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.
Summary
Background
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.
Methods
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.
Findings
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.
Interpretation
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.
Funding
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.
doi:10.1016/S0140-6736(10)62226-X
PMCID: PMC3789232  PMID: 21411136
children; heart rate; respiratory rate; normal; centiles; ranges
24.  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.
doi:10.1136/bcr.05.2011.4212
PMCID: PMC3166583  PMID: 22689275
25.  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.
doi:10.1016/j.imlet.2012.04.001
PMCID: PMC3407291  PMID: 22525013
T-cell Intracellular Antigen-1; allergen-mediated pulmonary inflammation; cytokines; translation

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