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2.  Evaluation of swabbing methods for estimating the prevalence of bacterial carriage in the upper respiratory tract: a cross sectional study 
BMJ Open  2014;4(10):e005341.
Bacterial carriage in the upper respiratory tract is usually asymptomatic but can lead to respiratory tract infection (RTI), meningitis and septicaemia. We aimed to provide a baseline measure of Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae and Neisseria meningitidis carriage within the community. Self-swabbing and healthcare professional (HCP) swabbing were compared.
Cross-sectional study.
Individuals registered at 20 general practitioner practices within the Wessex Primary Care Research Network South West, UK.
10 448 individuals were invited to participate; 5394 within a self-swabbing group and 5054 within a HCP swabbing group. Self-swabbing invitees included 2405 individuals aged 0–4 years and 3349 individuals aged ≥5 years. HCP swabbing invitees included 1908 individuals aged 0–4 years and 3146 individuals aged ≥5 years.
1574 (15.1%) individuals participated, 1260 (23.4%, 95% CI 22.3% to 24.5%) undertaking self-swabbing and 314 (6.2%, 95% CI 5.5% to 6.9%) undertaking HCP-led swabbing. Participation was lower in young children and more deprived practice locations. Swab positivity rates were 34.8% (95% CI 32.2% to 37.4%) for self-taken nose swabs (NS), 19% (95% CI 16.8% to 21.2%) for self-taken whole mouth swabs (WMS), 25.2% (95% CI 20.4% to 30%) for nasopharyngeal swabs (NPS) and 33.4% (95% CI 28.2% to 38.6%) for HCP-taken WMS. Carriage rates of S. aureus were highest in NS (21.3%). S. pneumoniae carriage was highest in NS (11%) and NPS (7.4%). M. catarrhalis carriage was highest in HCP-taken WMS (28.8%). H. influenzae and P. aeruginosa carriage were similar between swab types. N. meningitidis was not detected in any swab. Age and recent RTI affected carriage of S. pneumoniae and H. influenzae. Participant costs were lower for self-swabbing (£41.21) versus HCP swabbing (£69.66).
Higher participation and lower costs of self-swabbing as well as sensitivity of self-swabbing favour this method for use in large population-based respiratory carriage studies.
PMCID: PMC4216860  PMID: 25358677
3.  Perspectives on Tissue Interactions in Development and Disease 
Current molecular medicine  2010;10(1):95-112.
From the morphogenetic movements of the three germ layers during development to the reactive stromal microenvironment in cancer, tissue interactions are vital to maintaining healthy organ morphologic architecture and function. The stromal compartment is thought to be complicit in tumor progression and, as such, represents an opportune target for disease therapies. However, recent developments in our understanding of the diversity of the stromal compartment and the lack of appropriate models to study its relevance in human disease have limited our further understanding of the role of tissue interactions in tumor progression. The failure of any model to fully recapitulate the complexities of systemic biology continues to create a higher imperative for incorporating various perspectives into a broader understanding for the ultimate goal of designing interventional therapies. Understanding this potential, this review examines the biological models used to study stromal-epithelial interactions and includes an attempt to incorporate behavioral terminology to define and mathematically model ecological relationships in stromal-epithelial interactions. In addition, the current attempt to incorporate these diverse ecological perspectives into in silico mathematical models through cross-disciplinary coordination is reviewed, which will provide a fresh perspective on defining cell group behavior and tissue ecology in disease and hopefully lead to the generation of new hypotheses to be empirically validated.
PMCID: PMC4195241  PMID: 20205682
Tissue interaction; models; development; disease; stroma; epithelia; organ
4.  Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain 
Genome Announcements  2014;2(5):e00980-14.
Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity.
PMCID: PMC4183873  PMID: 25278529
5.  Paradoxical Adipose Hyperplasia After Cryolipolysis 
JAMA dermatology  2014;150(3):317-319.
Cryolipolysis is the non-invasive reduction of fat with localized cutaneous cooling. Since initial introduction, over 650,000 cryolipolysis treatment cycles have been performed worldwide. We present a previously unreported, rare side effect following cryolipolysis, paradoxical adipose hyperplasia.
A 41-year-old man underwent a single cycle of cryolipolysis to his abdomen. Three months following his treatment, a gradual enlargement of the treatment area was noted. This enlargement was a large, well-demarcated subcutaneous mass, slightly tender to palpation. Imaging studies revealed accumulation of adipose tissue with normal signal intensity within the treatment area.
Conclusions and Relevance
Paradoxical adipose hyperplasia is a rare, previously unreported side effect of cryolipolysis with an incidence of 0.0051%. No single unifying risk factor has been identified. The phenomenon seems to be more common in male patients undergoing cryolipolysis. At this time there is no evidence of spontaneous resolution. Further studies are needed to characterize the pathogenesis and histologic findings of this rare adverse event.
PMCID: PMC4171727  PMID: 24382640
6.  Influence of retinopathy on the achromatic and chromatic vision of patients with type 2 diabetes 
BMC Ophthalmology  2014;14:104.
Luminance contrast sensitivity and colour vision are considered to have great predictive value in the evaluation of type 2 diabetic retinopathy. However, these two visual characteristics have seldom been investigated in the same group of patients. In the present study we measured contrast sensitivity and colour vision in a group of patients with type 2 diabetes and correlated the results with estimates of common metabolic markers for the disease. A subgroup of the patients had no clinical signs of retinopathy.
The vision of 27 patients (n = 50 eyes) with type 2 diabetes, with retinopathy (n = 20 eyes), or without retinopathy (n = 30 eyes) were evaluated using two psychophysical tests, the Farnsworth–Munsell 100 hue test (FM 100), and measurements of the luminance contrast sensitivity at 11 spatial frequencies. The results were compared with measurements obtained from an age-matched control group (n = 32), and were correlated with the level of glycated haemoglobin, glycaemic level, and time of disease onset. Signs of retinopathy were identified during the ophthalmological examinations.
Contrast sensitivity and colour vision impairments were present at different levels in diabetes patients. Eyes with retinopathy showed more severe vision loss than eyes without retinopathy. The FM 100 test was more sensitive for separation of patients from controls. Colour vision loss had no colour axes preference. The contrast sensitivity test appeared to have some advantage in differentiating patients with retinopathy from patients without retinopathy.
Both methods can be useful to follow the visual function of diabetic patients and should be used together to discriminate patients from controls, as well as to identify early signs of retinal damage.
PMCID: PMC4236659  PMID: 25174264
Type 2 diabetes; Visual psychophysics; Diabetic retinopathy; Contrast sensitivity; Colour vision; Farnsworth–Munsell test
7.  Three-dimensional volumetric quantification of fat loss following cryolipolysis 
Lasers in surgery and medicine  2013;46(2):75-80.
Background and Objectives
Cryolipolysis is a noninvasive and well-tolerated treatment for reduction of localized subcutaneous fat. Although several studies demonstrate the safety and efficacy of this procedure, volumetric fat reduction from this treatment has not been quantified. This prospective study investigated the change in volume of fat after cryolipolysis treatment using three-dimensional (3D) photography.
Materials and Methods
A prospective study of subjects treated with cryolipolysis on the flank (love handle) was performed at Massachusetts General Hospital. Volume measurements were performed with a Canfield Scientific Vectra three-dimensional camera and software to evaluate the amount of post procedure volume change. Clinical outcomes were assessed with caliper measurements, subject surveys, and blinded physician assessment of photographs.
Eleven subjects were enrolled in this study. Each subject underwent a single cycle of cryolipolysis to one flank. The untreated flank served as an internal control. The follow up time after treatment was two months. The mean amount of calculated absolute fat volume loss using 3D photography from baseline to 2 months follow up visit was 56.2 ± 25.6 from the treatment site and 16.6 ± 17.6 cc from the control (p < 0.0001). A mean absolute difference of 39.6 cc between the treated and untreated sides was calculated at 2 months post-treatment. Comparison of caliper measurements from baseline to 2 months post-treatment demonstrated significant reduction of the treated flank from 45.6 ± 5.8 mm at baseline to 38.6 ±4.6 mm at 2 months post-treatment (p<0.001). The untreated flank did not show significant reduction with caliper measurements demonstrating 45.3 ± 5.0 mm at baseline and 44.6 ± 5.1 mm at 2 months post-treatment (p=0.360). No unexpected side effects or adverse events were reported. Post-treatment satisfaction surveys demonstrated 82% of subjects were satisfied with the results.
Cryolipolysis is a safe, well-tolerated and effective noninvasive fat removal methodology that on average leads to 39.6 cc of fat loss at 2 months after a single treatment cycle.
PMCID: PMC4123113  PMID: 24535759
fat removal; noninvasive; volume of fat; imaging; cold induced fat loss
8.  The prevalence of smoking and the knowledge of smoking hazards and smoking cessation strategies among HIV positive patients in Johannesburg, South Africa 
While the detrimental effects of smoking among HIV positive patients have been well documented, there is a paucity of data regarding cigarette smoking prevalence among these patients in South Africa.
To establish the frequency, demographics, and knowledge of harmful effects and of smoking cessation strategies among HIV-positive patients in Johannesburg, South Africa.
A prospective cross-sectional survey, using a structured questionnaire interview, of HIV-positive patients attending the HIV Clinic at the Charlotte Maxeke Johannesburg Academic Hospital between 1 July 2011 and 31 October 2011.
Of 207 HIV positive patients attending an ARV roll-out clinic, 31 (15%) were current smokers (23% of the males and 7.5% of the females) and a further 45 (21.7%) were ex-smokers. Most of the current smokers (30/31 patients) indicated their wish to quit smoking, and among the group as a whole most patients were aware of the general (82.5%) and HIV-related (77.8%) risks of smoking and of methods of quitting smoking. Despite this, however, most (61.8%) were not aware of whom they could approach for assistance and advice.
Given the relatively high prevalence of current and ex-smokers amongst HIV positive patients there is a need for the introduction of smoking cessation strategies and assistance at HIV-rollout clinics in South Africa.
PMCID: PMC4112726  PMID: 24148173
9.  Adverse Outcomes Among Women Presenting with Signs and Symptoms of Ischemia and No Obstructive Coronary Artery Disease: Findings from the NHLBI-sponsored Women’s Ischemia Syndrome Evaluation (WISE) Angiographic Core Laboratory 
American heart journal  2013;166(1):134-141.
Women presenting with signs and symptoms of myocardial ischemia frequently have no or non-obstructive coronary artery disease (CAD).
To investigate associations between angiographic measures and longer-term clinical outcomes among women with signs and symptoms of ischemia referred for coronary angiography.
Prospective cohort analysis of women referred for coronary angiography and enrolled in the National Heart, Lung, and Blood Institute-sponsored Women’s Ischemia Syndrome Evaluation (WISE). An angiographic severity score was prospectively developed, assigning points for any stenosis weighted by stenosis severity, location and collaterals, and then tested for prediction for adverse outcome in 917 women over a median 9.3 years.
Referral centers.
Women, with signs and/or symptoms of myocardial ischemia, referred for coronary angiography were consecutively consented and enrolled in a prospective study.
Main Outcome Measures
First occurrence of cardiovascular death or non-fatal myocardial infarction. Hospitalization for angina was a secondary outcome.
Cardiovascular death or myocardial infarction at 10 years occurred in 6.7%, 12.8% and 25.9% of women with no, non-obstructive, and obstructive CAD (p<0.0001), respectively. Cumulative 10-year cardiovascular death or MI rates showed progressive, near linear, increases for each WISE CAD severity score range of 5, 5.1–10, 10.1–20, 20.1–50, and >50. The optimal threshold in the WISE severity score classifications for predicting cardiovascular mortality was >10 (e.g. 5.0–10 vs. 10.1–89), with both a sensitivity and specificity of 0.64 and an area under the curve of 0.64 (p=0.02, 95% CI = 0.59, 0.68).
Among women with signs and symptoms of ischemia, non-obstructive CAD is common, and associated with adverse outcomes over the longer-term. The new WISE angiographic score appears to be useful for risk prediction in this population.
PMCID: PMC3703586  PMID: 23816032
angiography score; coronary disease; prognosis; women
10.  The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells 
Human Reproduction (Oxford, England)  2013;28(6):1635-1646.
What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster?
The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis.
The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report.
We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes.
We used quantitative reverse transcription–polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression.
We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes.
While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary.
The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.
This work was supported by the National Institutes of Health grants R01-HD053808 and -HD45595 (to M.F.W.), U54-HD012303 (to S.S. and R.J.C.), and K12-HD001259 (to L.C.L.), and Medical Research Council grant G1100357/1 (to R.A.A.). None of the authors declared a conflict of interest.
PMCID: PMC3657123  PMID: 23482336
testis; ovary; homeobox gene cluster; spermatogenesis; oogenesis
11.  Cysteinyl Leukotriene Receptor-1 Antagonists as Modulators of Innate Immune Cell Function 
Journal of Immunology Research  2014;2014:608930.
Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8+ cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5′-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.
PMCID: PMC4058211  PMID: 24971371
12.  TIMI Frame Count and Adverse Events in Women with No Obstructive Coronary Disease: A Pilot Study from the NHLBI-Sponsored Women's Ischemia Syndrome Evaluation (WISE) 
PLoS ONE  2014;9(5):e96630.
TIMI frame count (TFC) predicts outcomes in patients with obstructive coronary artery disease (CAD); it remains unclear whether TFC predicts outcomes in patients without obstructive CAD.
TFC was determined in a sample of women with no obstructive CAD enrolled in the Women's Ischemia Syndrome Evaluation (WISE) study. Because TFC is known to be higher in the left anterior descending artery (LAD), TFC determined in the LAD was divided by 1.7 to provide a corrected TFC (cTFC).
A total of 298 women, with angiograms suitable for TFC analysis and long-term (6–10 year) follow up data, were included in this sub-study. Their age was 55±11 years, most were white (86%), half had a history of smoking, and half had a history of hypertension. Higher resting cTFC was associated with a higher rate of hospitalization for angina (34% in women with a cTFC >35, 15% in women with a cTFC ≤35, P<0.001). cTFC provided independent prediction of hospitalization for angina after adjusting for many baseline characteristics. In this cohort, resting cTFC was not predictive of major events (myocardial infarction, heart failure, stroke, or all-cause death), cardiovascular events, all-cause mortality, or cardiovascular mortality.
In women with signs and symptoms of ischemia but no obstructive CAD, resting cTFC provides independent prediction of hospitalization for angina. Larger studies are required to determine if resting TFC is predictive of major events in patients without obstructive coronary artery disease.
PMCID: PMC4011756  PMID: 24800739
14.  Coronary Angiography: Is it Time to Reassess? 
Circulation  2013;127(17):1760-1762.
PMCID: PMC3746971  PMID: 23630085
angiography; stenosis; cardiovascular disease; diagnosis
15.  Evolving treatment strategies for colorectal cancer: A critical review of current therapeutic options 
Management of rectal cancer has markedly evolved over the last two decades. New technologies of staging have allowed a more precise definition of tumor extension. Refinements in surgical concepts and techniques have resulted in higher rates of sphincter preservation and better functional outcome for patients with this malignancy. Although, preoperative chemoradiotherapy followed by total mesorectal excision has become the standard of care for locally advanced tumors, many controversial matters in management of rectal cancer still need to be defined. These include the feasibility of a non-surgical approach after a favorable response to neoadjuvant therapy, the ideal margins of surgical resection for sphincter preservation and the adequacy of minimally invasive techniques of tumor resection. In this article, after an extensive search in PubMed and Embase databases, we critically review the current strategies and the most debatable matters in treatment of rectal cancer.
PMCID: PMC3921541  PMID: 24574762
Rectal cancer; Colorectal cancer; Staging; Sphincter preservation; Neoadjuvant chemo-radiotherapy; Surgery
16.  The ageing ovary and uterus: new biological insights 
Human Reproduction Update  2012;19(1):67-83.
Advanced maternal age is associated with reduced fertility and adverse pregnancy outcomes. This review details recent developments in our understanding of the biology and mechanisms underlying reproductive ageing in women and the implications for fertility and pregnancy.
Sociological online libraries (IBSS, SocINDEX), PubMed and Google Scholar were searched for relevant demographic, epidemiological, clinical and biological studies, using key words and hierarchical MeSH terms. From this, we identified and focused on key topics where it was judged that there had been clinically relevant advances in the understanding of ovarian and uterine ageing with implications for improved diagnostics and novel interventions.
Mapping of the ovarian reserve, follicular dynamics and associated biomarkers, across the reproductive lifespan has recently been performed. This now allows an assessment of the effects of environmental, lifestyle and prenatal exposures on follicular dynamics and the identification of their impact during periods of germ cell vulnerability and may also facilitate early identification of individuals with shorter reproductive lifespans. If women choose to time their family based on their ovarian reserve this would redefine the meaning of family planning. Despite recent reports of the potential existence of stem cells which may be used to restore the primordial follicle and thereby the oocyte pool, therapeutic interventions in female reproductive ageing at present remain limited. Maternal ageing has detrimental effects on decidual and placental development, which may be related to repeated exposure to sex steroids and underlie the association of ageing with adverse perinatal outcomes.
Ageing has incontrovertible detrimental effects on the ovary and the uterus. Our enhanced understanding of ovarian ageing will facilitate early identification of individuals at greatest risk, and novel therapeutic interventions. Changes in both ovary and uterus are in addition to age-related co-morbidities, which together have synergistic effects on reducing the probability of a successful pregnancy outcome.
PMCID: PMC3508627  PMID: 23103636
ageing; fertility; infertility; ovarian reserve; anti-Müllerian hormone
17.  Trends of the Incidence of Ischemic Stroke Thrombolysis over Seven Years and One-Year Outcome: A Population-Based Study in Joinville, Brazil 
Cerebrovascular Diseases Extra  2013;3(1):156-166.
In a population-based setting, we aimed to measure the incidence trends of ischemic stroke (IS) thrombolysis, thrombolysis times, proportion of symptomatic intracerebral hemorrhage (sICH), 30-day case fatality and functional outcomes. We also compared the 12-month functional status between thrombolyzed and nonthrombolyzed patients.
Using data from the Joinville Population-Based Stroke Registry, we prospectively ascertained a cohort of all thrombolyses done in Joinville citizens, Southern Brazil, from 2005 to 2011. For the definition of sICH we used European Cooperative Acute Stroke Study (ECASS) II criteria.
Over 7 years, 6% (220/3,552) of all IS were thrombolyzed. The thrombolysis incidence increased from 1.4 [95% confidence interval (CI), 0.6-2.9] in 2005 to 9.8 (7.3-12.9) per 100,000 population in 2011 (p < 0.0001). The thrombolysis incidence age-adjusted to the world population in 2011 was 11 (8.2-14.3) per 100,000. Only 30% (50/165) were thrombolyzed within 1 h of arrival at hospital. In 7 days, 6.4% (14/220) had sICH and 57% (8/14) of those died. In the 2009-2011 period, a favorable functional outcome [modified Rankin scale (mRS) 0-1] at 12 months among patients who received thrombolysis was more frequent [mRS 0-1; 36% (38/107)] than among patients who did not receive thrombolysis [mRS 0-1; 24% (131/544); p = 0.016]. The logistic regression showed that thrombolyzed IS patients had a more favorable outcome (mRS 0-1; HR 2.13; 95% CI, 1.2-3.7; p < 0.016) than nonthrombolyzed patients.
In a population setting of a middle income country, the thrombolysis incidence and outcomes were similar to those of other well-structured services. After 1 year, patients thrombolyzed in the 4.5-hour time window had a better outcome. More than proportions, rates provide additional information and could be used to benchmark services against others.
PMCID: PMC3924708  PMID: 24570681
Ischemic stroke; Thrombolysis; Population-based study; Incidence; Outcome; Middle income country; Epidemiology

18.  Mature Epitope Density - A strategy for target selection based on immunoinformatics and exported prokaryotic proteins 
BMC Genomics  2013;14(Suppl 6):S4.
Current immunological bioinformatic approaches focus on the prediction of allele-specific epitopes capable of triggering immunogenic activity. The prediction of major histocompatibility complex (MHC) class I epitopes is well studied, and various software solutions exist for this purpose. However, currently available tools do not account for the concentration of epitope products in the mature protein product and its relation to the reliability of target selection.
We developed a computational strategy based on measuring the epitope's concentration in the mature protein, called Mature Epitope Density (MED). Our method, though simple, is capable of identifying promising vaccine targets. Our online software implementation provides a computationally light and reliable analysis of bacterial exoproteins and their potential for vaccines or diagnosis projects against pathogenic organisms. We evaluated our computational approach by using the Mycobacterium tuberculosis (Mtb) H37Rv exoproteome as a gold standard model. A literature search was carried out on 60 out of 553 Mtb's predicted exoproteins, looking for previous experimental evidence concerning their possible antigenicity. Half of the 60 proteins were classified as highest scored by the MED statistic, while the other half were classified as lowest scored. Among the lowest scored proteins, ~13% were confirmed as not related to antigenicity or not contributing to the bacterial pathogenicity, and 70% of the highest scored proteins were confirmed as related. There was no experimental evidence of antigenic or pathogenic contributions for three of the highest MED-scored Mtb proteins. Hence, these three proteins could represent novel putative vaccine and drug targets for Mtb. A web version of MED is publicly available online at
The software presented here offers a practical and accurate method to identify potential vaccine and diagnosis candidates against pathogenic bacteria by "reading" results from well-established reverse vaccinology software in a novel way, considering the epitope's concentration in the mature portion of the protein.
PMCID: PMC3908659  PMID: 24564223
19.  The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence 
Do the ovarian follicles of children and adolescents differ in their morphology and in vitro growth potential from those of adults?
Pre-pubertal ovaries contained a high proportion of morphologically abnormal non-growing follicles, and follicles showed reduced capacity for in vitro growth.
The pre-pubertal ovary is known to contain follicles at the early growing stages. How this changes over childhood and through puberty is unknown, and there are no previous data on the in vitro growth potential of follicles from pre-pubertal and pubertal girls.
Ovarian biopsies from five pre-pubertal and seven pubertal girls and 19 adult women were analysed histologically, cultured in vitro for 6 days, with growing follicles then isolated and cultured for a further 6 days.
Ovarian biopsies were obtained from girls undergoing ovarian tissue cryopreservation for fertility preservation, and compared with biopsies from adult women. Follicle stage and morphology were classified. After 6 days in culture, follicle growth initiation was assessed. The growth of isolated secondary follicles was assessed over a further 6 days, including analysis of oocyte growth.
Pre-pubertal ovaries contained a high proportion of abnormal non-growing follicles (19.4 versus 4.85% in pubertal ovaries; 4004 follicles analysed; P = 0.02) characterized by indistinct germinal vesicle membrane and absent nucleolus. Follicles with this abnormal morphology were not seen in the adult ovary. During 6 days culture, follicle growth initiation was observed at all ages; in pre-pubertal samples there was very little development to secondary stages, while pubertal samples showed similar growth activation to that seen in adult tissue (pubertal group: P = 0.02 versus pre-pubertal, ns versus adult). Isolated secondary follicles were cultured for a further 6 days. Those from pre-pubertal ovary showed limited growth (P < 0.05 versus both pubertal and adult follicles) and no change in oocyte diameter over that period. Follicles from pubertal ovaries showed increased growth; this was still reduced compared with follicles from adult women (P < 0.05) but oocyte growth was proportionate to follicle size.
These data derive from only a small number of ovarian biopsies, although large numbers of follicles were analysed. It is unclear whether the differences between groups are related to puberty, or just age.
These findings show that follicles from girls of all ages can be induced to grow in vitro, which has important implications for some patients who are at high risk of malignant contamination of their ovarian tissue. The reduced growth of isolated follicles indicates that there are true intrafollicular differences in addition to potential differences in their local environment, and that there are maturational processes occurring in the ovary through childhood and adolescence, which involve the loss of abnormal follicles, and increasing follicle developmental competence.
Study funding/competing interest(s)
Funded by MRC grants G0901839 and G1100357. No competing interests.
PMCID: PMC3860895  PMID: 24135076
childhood; follicle; ovary; puberty; adolescence
20.  Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity 
Conventional autologous skin grafts are associated with significant donor-site morbidity. This study was conducted to determine feasibility, safety, and efficacy of a new strategy for skin grafting based on harvesting small columns of full-thickness skin with minimal donor-site morbidity.
The swine model was used for this study. Hundreds of full-thickness columns of skin tissue (~700 µm diameter) were harvested using a custom-made harvesting device, and then applied directly to excisional skin wounds. Healing in donor and graft sites was evaluated over 3 months by digital photographic measurement of wound size and blinded, computer-aided evaluation of histological features and compared with control wounds that healed by secondary intention or with conventional split-thickness skin grafts (STSG).
After harvesting hundreds of skin columns, the donor sites healed rapidly without scarring. These sites reepithelialized within days and were grossly and histologically indistinguishable from normal skin within 7 weeks. By contrast, STSG donor sites required 2 weeks for reepithelialization and retained scar-like characteristics in epidermal and dermal architecture throughout the experiment. Wounds grafted with skin columns resulted in accelerated reepithelialization compared with ungrafted wounds while avoiding the “fish-net” patterning caused by STSG.
Full-thickness columns of skin can be harvested in large quantities with negligible long-term donor-site morbidity, and these columns can be applied directly to skin wounds to enhance wound healing.
PMCID: PMC4174164  PMID: 25289241
21.  Effect of the Use and Timing of Bone Marrow Mononuclear Cell Delivery on Left Ventricular Function After Acute Myocardial Infarction: The TIME Randomized Trial 
While the delivery of cell therapy following ST segment myocardial infarction (STEMI) has been evaluated in previous clinical trials, the influence of the timing of cell delivery on the effect on left ventricular (LV) function has not been analyzed in a trial that randomly designated the time of delivery.
To determine 1) the effect of intracoronary autologous bone marrow mononuclear cell (BMC) delivery following STEMI on recovery of global and regional LV function and 2) if timing of BMC delivery (3 versus 7 days following reperfusion) influences this effect.
Design, Setting, and Patients
Between July 17, 2008 and November 15, 2011, 120 patients were enrolled in a randomized, 2×2 factorial, double-blind, placebo-controlled trial of the National Heart, Lung, and Blood Institute (NHLBI)-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) of patients with LV dysfunction (LV Ejection Fraction (LVEF) ≤45%) following successful primary percutaneous coronary intervention (PCI) of anterior STEMI.
Intracoronary infusion of 150 × 106 BMCs or placebo (randomized 2:1 BMC:placebo) within 12 hours of aspiration and processing administered at Day 3 or Day 7 (randomized 1:1) post-PCI.
Main Outcome Measures
Co-primary endpoints were: 1) Change in global (LVEF) and regional (wall motion) LV function in infarct and border zones at 6 months measured by cardiac magnetic resonance imaging and 2) Change in LV function as affected by timing of treatment on Day 3 versus Day 7. Secondary endpoints included major adverse cardiovascular events as well as changes in LV volumes and infarct size.
Patient mean age was 56.9±10.9 years with 87.5% male. At 6 months, LVEF increased similarly in both BMC (45.2±10.6 to 48.3±13.3 %) and placebo groups (44.5±10.8 to 47.8±13.6 %). No detectable treatment effect on regional LV function was observed in either infarct or border zones. Differences between therapy groups in the change in global LV function over time when treated at Day 3 (−0.9±2.9%, 95% CI 6.6 to 4.9%, p=0.763) or Day 7 (1.1±2.9%, 95% CI −4.7 to 6.9, p=0.702) were not significant, nor were they different from each other. Also, timing of treatment had no detectable effect on recovery of regional LV function. Major adverse events were rare with no difference between groups.
Patients with STEMI, who underwent successful primary PCI and administration of intra-coronary BMCs at either 3 or 7 days following the event, had recovery of global and regional LV function similar to placebo
Trial Registration Number, NCT00684021
PMCID: PMC3652242  PMID: 23129008
22.  Safety of Coronary Reactivity Testing in Women with No Obstructive Coronary Artery Disease: Results from the NHLBI-sponsored Women’s Ischemia Syndrome Evaluation (WISE) Study 
We evaluated the safety of coronary reactivity testing (CRT) in symptomatic women with evidence of myocardial ischemia and no obstructive coronary artery disease (CAD).
Microvascular coronary dysfunction (MCD) in women with no obstructive CAD portends an adverse prognosis of 2.5% annual major adverse cardiovascular event (MACE) rate. The diagnosis of MCD is established by invasive CRT, yet the risk of CRT is unknown.
We evaluated 293 symptomatic women with ischemia and no obstructive CAD, who underwent CRT at three experienced centers. Microvascular function was assessed using a Doppler wire and injections of adenosine, acetylcholine, and nitroglycerin in the left coronary artery. CRT-related serious adverse events (SAE), adverse events (AE), and follow-up MACE (death, nonfatal myocardial infarction (MI), nonfatal stroke, or hospitalization for heart failure) were recorded.
CRT-SAEs occurred in 2 women (0.7%) during the procedure: one had coronary artery dissection, and one developed MI associated with coronary spasm. CRT-AEs occurred in 2 women (0.7%) and included one transient air microembolism and one deep venous thrombosis. There was no CRT-related mortality. In the mean follow-up period of 5.4 years, the MACE rate was 8.2%, including 5 deaths (1.7%), 8 non-fatal MIs (2.7%), 8 nonfatal strokes (2.7%), and 11 hospitalizations for heart failure (3.8%).
In women undergoing CRT for suspected MCD, contemporary testing carries a relatively low risk compared to the MACE rate in these women. These results support the use of CRT by experienced operators for establishing definitive diagnosis and assessing prognosis in this at-risk population.
PMCID: PMC3417766  PMID: 22721660
coronary reactivity; microvascular dysfunction; endothelial dysfunction
23.  Keep a way open for tailored treatments 
Nature  2012;484(7394):318.
PMCID: PMC3375895  PMID: 22517151
24.  Effect of Transendocardial Delivery of Autologous Bone Marrow Mononuclear Cells on Functional Capacity, Left Ventricular Function, and Perfusion in Chronic Ischemic Heart Failure: The FOCUS-CCTRN Trial 
Previous studies utilizing autologous bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy have demonstrated safety and suggested efficacy. The FOCUS protocol was designed to assess efficacy of a larger cell dose in an adequately well-powered phase II study.
To determine if administration of BMCs through transendocardial injections improves myocardial perfusion, reduces left ventricular (LV) end systolic volume, or enhances maximal oxygen consumption in patients with coronary artery disease (CAD), LV dysfunction, and limiting heart failure and/or angina.
Design, Setting, and Patients
This is a 100 million cell, first-in-man randomized, double-blind, placebo-controlled trial was performed by the National Heart, Lung, and Blood Institute-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) in symptomatic patients (NYHA II-III and/or CCS II-IV) receiving maximal medical therapy, with a left ventricular ejection fraction (LVEF)≤45%, perfusion defect by single-photon emission tomography (SPECT), and CAD not amenable to revascularization.
All patients underwent bone marrow aspiration, isolation of BMCs using a standardized automated system performed locally, and transendocardial injection of 100 million BMCs or placebo (2:1 BMC: placebo).
Main Outcome Measures
Three co-primary endpoints assessed at 6 months were changes in (a) LV end systolic volume (LVESV) by echocardiography, (b) maximal oxygen consumption (MVO2), and (c) reversibility on SPECT. Secondary measures included other SPECT measures, magnetic resonance imaging (MRI), echocardiography, clinical improvement, and major adverse cardiac events (MACE). Phenotypic and functional analyses of the cell product were performed by the CCTRN Biorepository lab.
Of 153 consented patients, a total of 92 (82 men; average age, 63 years) were randomized (n= 61 BMC, 31 placebo) at 5 sites between April 29, 2009 and April 18, 2011. Changes in LVESV index, (−0.9 ± 11.3 mL/m2; P = 0.733; 95% CI, −6.1 to 4.3), MVO2 (1.0 ± 2.9; P = 0.169; 95% CI, −0.42 to 2.34), percent reversible defect change, (−1.2 ± 23.3; P = 0.835; 95% CI, −12.50 to 10.12), and incidence of MACEwere not statistically significant. However, in an exploratory analysis the change in LVEF across the entire cohort by therapy group was significant (2.7 ± 5.2%; P = 0.030; 95% CI, 0.27 to 5.07).
This is the largest cell therapy trial of autologous BMCs in patients with ischemic LV dysfunction. In patients with chronic ischemic heart disease, transendocardial injection of BMCs compared to placebo did not improve LVESV, MVO2, or reversibility on SPECT.
PMCID: PMC3600947  PMID: 22447880
Chronic CAD; Ischemic Heart Failure; Chronic Angina; bone marrow mononuclear cells; cardiac performance
25.  Excitatory Amino Acid Transporter 5 is widely expressed in peripheral tissues 
It is routinely stated in the literature that Excitatory Amino Acid Transporter 5 (EAAT5) is a retina-specific glutamate transporter. EAAT5 is expressed by retinal photoreceptors and bipolar cells, where it serves as a slow transporter and as an inhibitory glutamate receptor, the latter role is due to the gating of a large chloride conductance. The dogma of an exclusively retinal distribution has arisen because Northern blot analyses have previously shown only modest hybridisation in non-retinal tissues. Others have re-interpreted this as indicating that EAAT5 was only present in retinal tissues. However, this view appears to be erroneous; recent evidence demonstrating abundant expression of EAAT5 in rat testis prompted us to re-examine this dogma. A new antibody was developed to an intracellular loop region of rat EAAT5. This new tool, in concert with RT-PCR and sequencing, demonstrated that EAAT5 is widely distributed at the mRNA and protein levels in many non-nervous tissues including liver, kidney, intestine, heart, lung, and skeletal muscle. We conclude that EAAT5 is a widely distributed protein. Whether it functions in all locations as a glutamate transporter, or mainly as a glutamate-gated chloride conductance, remains to be determined.
PMCID: PMC3683608  PMID: 23549460
EAAT5; glutamate; transporter; heart; lung; kidney.

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