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1.  Therapeutic Inflammatory Monocyte Modulation Using Immune-Modifying Microparticles 
Science translational medicine  2014;6(219):219ra7.
Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate–induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.
PMCID: PMC3973033  PMID: 24431111
2.  Human TH9 cells are skin-tropic and have autocrine and paracrine pro-inflammatory capacity 
Science translational medicine  2014;6(219):219ra8.
T helper type 9 (TH9) cells can mediate tumor immunity and participate in autoimmune and allergic inflammation in mice but little is known about the TH9 cells that develop in vivo in humans. We isolated T cells from human blood and tissues and found that most memory TH9 cells were skin-tropic or skin-resident. Human TH9 cells co-expressed TNFα and granzyme B, lacked coproduction of TH1/TH2/TH17 cytokines and many were specific for C. albicans. IL-9 production was transient and preceded the up-regulation of other inflammatory cytokines. Blocking studies demonstrated that IL-9 was required for maximal production of IFN-γ, IL-9, IL-13, and IL-17 by skin tropic T cells. IL-9 producing T cells were increased in the skin lesions of psoriasis, suggesting these cells may contribute to human inflammatory skin disease. Our results indicate human TH9 cells are a discrete T cell subset, many are tropic for the skin, and although they may function normally to protect against extracellular pathogens, aberrant activation of these cells may contribute to inflammatory diseases of the skin.
PMCID: PMC4102325  PMID: 24431112
3.  Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity 
Study question
S-Nitrosoglutathione is an endogenous airway smooth muscle relaxant. Increased airway S-Nitrosoglutathione breakdown occurs in some asthma patients. We asked whether patients with increased airway catabolism of this molecule had clinical features that distinguished them from other asthma patients.
We measured S-Nitrosoglutathione reductase expression and activity in bronchoscopy samples from 66 subjects in the Severe Asthma Research Program. We also analysed phenotype and genotype data from in the program as a whole.
Airway S-nitrosoglutathione reductase activity was increased in asthma patients (p = 0.032). However, only a subpopulation was affected, and this subpopulation was not defined by a “severe asthma” diagnosis. Subjects with increased activity were younger, had higher Immunoglobulin E and an earlier onset of symptoms. Consistent with a link between S-Nitrosoglutathione biochemistry and atopy, 1) interleukin 13 increased S-Nitrosoglutathione reductase expression, and 2) subjects with an S-Nitrosoglutathione reductase single nucleotide polymorphism (SNP) previously associated asthma had higher Immunoglobulin E than those without this SNP. Expression was higher in airway epithelium than in smooth muscle, and was increased in regions of the asthmatic lung with decreased airflow.
Answer to the question
An early-onset, allergic phenotype characterizes the asthma population with increased S-Nitrosoglutathione reductase activity.
PMCID: PMC4283933  PMID: 25359343
4.  FValidation and Psychometric Properties of the Asthma Control Questionnaire among Children 
The Journal of allergy and clinical immunology  2013;133(1):10.1016/j.jaci.2013.06.029.
The Asthma Control Questionnaire (ACQ) is a patient-centered tool for evaluating asthma control. It has been validated in adults but not well-validated among children.
We evaluated the reliability, validity, and responsiveness to change of the ACQ for assessing asthma control in children ages 6 to 17. A threshold value for poor disease control and a minimally important difference (MID) were also determined.
Data from 305 asthmatic children enrolled in a clinical trial were examined. The ACQ was administered at 8 visits. We analyzed results for the combined age group and for age groups 6 to 11 and 12 to 17 separately.
Overall, the Cronbach’s alpha (internal consistency) for the ACQ was 0.74 at baseline, and the intraclass correlation coefficient (test-retest reliability) for repeated questionnaires among stable patients was 0.53. The Pearson correlations between the ACQ and other asthma questionnaires were moderate to strong (−0.64 to −0.73). Mean ACQ scores were higher (worse) in patients whose peak flow decreased, who used more rescue medications, or who sought medical care for asthma than in patients who were stable (P<0.0001 for all measures). Change in ACQ scores were significantly different among patients with deteriorating, improving, or stable asthma symptoms (P ≤0.01). The optimal threshold indicating poor asthma control was ≥1.25. The MID was established to be 0.40. Results for the separate age groups were similar.
The ACQ is a moderately reliable, valid, and responsive tool with adequate psychometric properties for assessing recent asthma control among children.
PMCID: PMC3874246  PMID: 23932458
asthma; asthma control; Asthma Control Questionnaire; validity; minimally important difference
5.  Tracking Post-Hibernation Behavior and Early Migration Does Not Reveal the Expected Sex-Differences in a “Female-Migrating” Bat 
PLoS ONE  2014;9(12):e114810.
Long-distance migration is a rare phenomenon in European bats. Genetic analyses and banding studies show that females can cover distances of up to 1,600 km, whereas males are sedentary or migrate only short distances. The onset of this sex-biased migration is supposed to occur shortly after rousing from hibernation and when the females are already pregnant. We therefore predicted that the sexes are exposed to different energetic pressures in early spring, and this should be reflected in their behavior and physiology. We investigated this in one of the three Central European long-distance migrants, the common noctule (Nyctalus noctula) in Southern Germany recording the first individual partial migration tracks of this species. In contrast to our predictions, we found no difference between male and female home range size, activity, habitat use or diet. Males and females emerged from hibernation in similar body condition and mass increase rate was the same in males and females. We followed the first migration steps, up to 475 km, of radio-tagged individuals from an airplane. All females, as well as some of the males, migrated away from the wintering area in the same northeasterly direction. Sex differences in long-distance migratory behavior were confirmed through stable isotope analysis of hair, which showed greater variation in females than in males. We hypothesize that both sexes faced similarly good conditions after hibernation and fattened at maximum rates, thus showing no differences in their local behavior. Interesting results that warrant further investigation are the better initial condition of the females and the highly consistent direction of the first migratory step in this population as summering habitats of the common noctule occur at a broad range in Northern Europe. Only research focused on individual strategies will allow us to fully understand the migratory behavior of European bats.
PMCID: PMC4269398  PMID: 25517947
Angiology  2013;10.1177/0003319713489769.
We determined the association between daily ambulatory activity and markers of inflammation and oxidative stress in patients with peripheral artery disease (PAD) and claudication. Patients with PAD (n=134) limited by claudication were studied. Patients took 3275±1743 daily strides during 273±112 min each day, and their average daily cadence was 11.7±2.7 strides/min. High-sensitivity C-reactive protein was significantly and negatively associated with the total number of daily strides (p<0.001), total daily ambulatory time (p<0.01), peak activity index (p<0.01), daily average cadence (p<0.05), and the maximum cadences for 60 min (p<0.05), 30 min (p<0.05), 20 min (p<0.05) and 5 min (p<0.01). Oxidized low density lipoprotein and soluble vascular cell adhesion molecule-1 were not significantly associated with any of the ambulatory measures (p>0.05). We conclude that higher levels of community-based, daily ambulatory activity are associated with lower levels of inflammation, but are not associated with markers of oxidative stress.
PMCID: PMC3841224  PMID: 23695338
Claudication; C-Reactive Protein; Oxidized Low Density Lipoprotein; Peripheral Artery Disease
7.  Neuropilin-1 Expression Is Induced on Tolerant Self-Reactive CD8+ T Cells but Is Dispensable for the Tolerant Phenotype 
PLoS ONE  2014;9(10):e110707.
Establishing peripheral CD8+ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8+ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8+ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4+ and CD8+ T cells. Despite high expression on tolerant CD8+ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.
PMCID: PMC4208794  PMID: 25343644
8.  Recurrent PTPRB and PLCG1 mutations in angiosarcoma 
Nature genetics  2014;46(4):376-379.
Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionising radiation or chronic lymphoedema1. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signalling genes, as a key driver of angiosarcoma1. Here, we employed whole genome, exome, and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harboured predominantly truncating mutations in 10/39 (26%) tumours. PLCG1, a signal transducer of tyrosine kinases, presented with a recurrent, likely activating R707Q missense variant in 3/34 cases (9%). Overall, 15/39 (38%) tumours harboured at least one driver mutation in angiogenesis signalling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signalling in angiosarcoma.
PMCID: PMC4032873  PMID: 24633157
9.  Polygenic in vivo validation of cancer mutations using transposons 
Genome Biology  2014;15(9):455.
The in vivo validation of cancer mutations and genes identified in cancer genomics is resource-intensive because of the low throughput of animal experiments. We describe a mouse model that allows multiple cancer mutations to be validated in each animal line. Animal lines are generated with multiple candidate cancer mutations using transposons. The candidate cancer genes are tagged and randomly expressed in somatic cells, allowing easy identification of the cancer genes involved in the generated tumours. This system presents a useful, generalised and efficient means for animal validation of cancer genes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0455-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4210617  PMID: 25260652
10.  Randomized Clinical Trial of Lansoprazole for Poorly Controlled Asthma in Children 
Asymptomatic gastroesophageal reflux (GER) is prevalent in children with asthma. It is not known whether treatment of GER with a proton-pump inhibitor (PPI) improves asthma control.
To determine whether lansoprazole is effective in reducing asthma symptoms in children without overt GER.
Design, Setting, and Patients
A multicenter, randomized, masked, placebo controlled, parallel clinical trial comparing lansoprazole to placebo in children with poor asthma control on inhaled corticosteroid treatment conducted at 18 academic clinical centers. Participants were followed for 24 weeks. A subgroup had an esophageal pH study before randomization.
Children received either lansoprazole (15 mg daily < 30 kg; 30 mg ≥ 30 kg) or placebo, 1:1 allocation ratio.
Main outcome
The primary outcome was the change in Asthma Control score (ACQ, range from 0 to 6). Secondary outcomes included lung function measures, asthma-related quality of life and acute episodes of poor asthma control.
306 children were enrolled from April 2011 to August 2010, the median age was 11. The mean change (95% confidence interval (CI)) in the ACQ score was −0.1 (−0.2, 0.1) and −0.2 (−0.4, −0.1) units for the lansoprazole and placebo groups, respectively (P=0.12). There were no detectable treatment differences in secondary outcomes (mean (95% CI) for FEV1(0.00 (−0.08, 0.08)), asthma quality of life (−0.1 (−0.4, 0.1) or episodes of poor asthma control, hazard ratio of 1.18 (95% CI 0.91, 1.53). Among the 115 children with esophageal pH studies, the prevalence of GER was 43%. In the subgroup with a positive pH study, no treatment effect for lansoprazole versus placebo was observed for any asthma outcome. Children treated with lansoprazole reported more upper respiratory infections (63% vs 49%, P=0.02), sore throats (52% vs 39%, P=0.02), and bronchitis (7% vs 2%, P=0.05).
Among children with poorly controlled asthma without symptoms of GER who were using inhaled corticosteroids, the addition of lansoprazole, as compared to placebo, did not improve symptoms nor lung function but was associated with increased adverse events.
PMCID: PMC4153372  PMID: 22274684
11.  Obesity in Children with Poorly-Controlled Asthma: Sex Differences 
Pediatric pulmonology  2012;48(9):847-856.
Obesity increases asthma risk, and may alter asthma severity. In adults, sex appears to modify the effect of obesity on asthma. Among children, the effect of sex on the relationship between obesity and asthma severity remains less clear, particularly when considering race.
To determine how obesity affects disease characteristics in a diverse cohort of children with poorly controlled asthma, and if obesity effects are altered by sex.
We analyzed 306 children between 6–17 years of age with poorly controlled asthma enrolled in a 6-month trial assessing lansoprazole for asthma control. In this secondary analysis, we determined associations between obesity and symptom severity, spirometry, exacerbation risk, airway biomarkers, bronchial reactivity and airflow perception. We used both a multivariate linear regression and longitudinal mixed-effect model to determine if obesity interacted with sex to affect asthma severity.
Regardless of sex, BMI>95th percentile did not affect asthma control, exacerbation risk or airway biomarkers. Sex changed the effect of obesity on lung function (sex*obesity FEV1%, interaction p-value <.01, sex*obesity FEV1/FVC, interaction p-value=.03). Obese males had significantly worse airflow obstruction compared to non-obese males, while in females there was no obesity effect on airflow obstruction. In females, obesity was associated with significantly greater FEV1 and FVC, and a trend toward reduced airway reactivity.
Obesity did not affect asthma control, airway markers or disease stability; however obesity did affect lung function in a sex-dependent manner. In males, obesity associated with reduced FEV1/FVC, and in females, obesity associated with substantially improved lung function.
PMCID: PMC3578966  PMID: 23143849
Asthma; Obesity; Children; Sex; Body Mass Index; Spirometry
12.  fMRI Assessment of Thalamocortical Connectivity during Attentional Performance 
Magnetic resonance imaging  2013;31(7):1112-1118.
Functional magnetic resonance imaging (fMRI) studies have shown dysfunction in key areas associated with the thalamocortical circuit in patients with schizophrenia. This study examined the functional connectivity involving the frontal-thalamic circuitry during a spatial focusing-of-attention task in 18 unmedicated patients with schizophrenia and 38 healthy controls. Functional connectivity was analyzed by assigning seed regions (in the thalamic nuclei (mediodorsal nucleus (MDN), pulvinar, anterior nucleus (AN)), the dorsolateral prefrontal cortex (Brodmann areas 9 and 46), and the caudate), and correlating their respective activity with that in the non-seed regions voxel-wise. Functional connectivity analysis demonstrated that functional connectivity was significantly impaired in patients e.g., between the right pulvinar and regions such as the prefrontal and temporal cortices and the cerebellum. On the other hand, enhanced functional connectivity was found in patients e.g., between the AN and regions such as the prefrontal and temporal cortices. In addition, the patients had significantly lower task performance and less (but non-significant) brain activation than those of controls. These results revealed disturbed functional integration in schizophrenia, and suggested that the functional connectivity abnormalities in the thalamocortical circuitry, especially the frontal-thalamic circuitry, may underlie the attention deficits in schizophrenia patients. Further, this study suggested that functional connectivity analysis might be more sensitive than brain activation analysis in detecting the functional abnormalities in schizophrenia.
PMCID: PMC3729917  PMID: 23727467
Thalamocortical circuitry; Functional connectivity; Spatial attention
13.  Telemedicine Collaboration Improves Perinatal Regionalization and Lowers Statewide Infant Mortality 
We assessed a telemedicine (TM) network's effects on decreasing deliveries of very low birth-weight (VLBW, <1500 grams) neonates in hospitals without Neonatal Intensive Care Units (NICUs) and statewide infant mortality.
This prospective study used obstetrical and neonatal interventions through TM consults, education, and census rounds with 9 hospitals from July 1, 2009 – March 31, 2010. Using a generalized linear model, Medicaid data compared VLBW birth sites, mortality, and morbidity before and after TM use. Arkansas Health Department data and chi square analysis were used to compare infant mortality.
Deliveries of VLBW neonates in targeted hospitals decreased from 13.1% to 7.0% (p=0.0099); deliveries of VLBW neonates in remaining hospitals was unchanged. Mortality decreased in targeted hospitals (13.0% before TM and 6.7% after TM). Statewide infant mortality decreased from 8.5 to 7.0 per 1000 deliveries (p=0.043).
TM decreased deliveries of VLBW neonates in hospitals without NICUs and was associated with decreased statewide infant mortality..
PMCID: PMC4138978  PMID: 23579490
Very Low Birth Weight; Premature; Neonates; Neonatal; Neonatal Intensive Care Unit
14.  In Vitro Induced Regulatory T Cells Are Unique from Endogenous Regulatory T Cells and Effective at Suppressing Late Stages of Ongoing Autoimmunity 
PLoS ONE  2014;9(8):e104698.
Strategies to boost the numbers and functions of regulatory T cells (Tregs) are currently being tested as means to treat autoimmunity. While Tregs have been shown to be effective in this role, strategies to manipulate Tregs to effectively suppress later stages of ongoing diseases need to be established. In this study, we evaluated the ability of TGF-β-induced Tregs (iTregs) specific for the major self-antigen in autoimmune gastritis to suppress established autoimmune gastritis in mice. When transferred into mice during later stages of disease, iTregs demethylated the Foxp3 promoter, maintained Foxp3 expression, and suppressed effector T cell proliferation. More importantly, these iTregs were effective at stopping disease progression. Untreated mice had high numbers of endogenous Tregs (enTregs) but these were unable to stop disease progression. In contrast, iTregs, were found in relatively low numbers in treated mice, yet were effective at stopping disease progression, suggesting qualitative differences in suppressor functions. We identified several inhibitory receptors (LAG-3, PD-1, GARP, and TNFR2), cytokines (TGF-β1 and IL12p35), and transcription factors (IRF4 and Tbet) expressed at higher levels by iTregs compared to enTregs isolated form mice with ongoing disease, which likely accounts for superior suppressor ability in this disease model. These data support efforts to use iTregs in therapies to treat establish autoimmunity, and show that iTregs are more effective than enTregs at suppressing inflammation in this disease model.
PMCID: PMC4131893  PMID: 25119105
16.  Th2 cytokines from malignant cells suppress Th1 responses and enforce a global Th2 bias in leukemic cutaneous T cell lymphoma 
In leukemic CTCL (L-CTCL) malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased IgE, decreased Th1 responses and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, Th2 and Th17 cells.
Experimental design
We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells and studied the immunomodulatory effects of treatment modalities in L-CTCL patients.
12/12 L-CTCL patients overproduced Th2 cytokines. Remaining benign T cells were also strongly Th2 biased, suggesting a global Th2 skewing of the T cell repertoire. Culture of benign T cells away from the malignant clone reduced Th2 and enhanced Th1 responses but separate culture had no effect on malignant T cells. Co-culture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to IL-4 and IL-13 restored Th1 responses. In patients, enhanced Th1 responses were observed following a variety of treatment modalities that reduced malignant T cell burden.
A global Th2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. Th2 cytokines from malignant cells strongly inhibited Th1 responses. Our results suggest therapies that inhibit Th2 cytokine activity, by virtue of their ability to improve Th1 responses, may have the potential to enhance both anti-cancer and anti-pathogen responses.
PMCID: PMC3715586  PMID: 23785046
17.  Intramuscular Therapeutic Vaccination Targeting HPV16 Induces T Cell Responses That Localize in Mucosal Lesions 
Science translational medicine  2014;6(221):221ra13.
About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8+ T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNβ), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation.
PMCID: PMC4086631  PMID: 24477000
18.  Inflammation and Neurological Disease-Related Genes are Differentially Expressed in Depressed Patients with Mood Disorders and Correlate with Morphometric and Functional Imaging Abnormalities 
Brain, behavior, and immunity  2012;31:161-171.
Depressed patients show evidence of both proinflammatory changes and neurophysiological abnormalities such as increased amygdala reactivity and volumetric decreases of the hippocampus and ventromedial prefrontal cortex (vmPFC). However, very little is known about the relationship between inflammation and neuroimaging abnormalities in mood disorders. A whole genome expression analysis of peripheral blood mononuclear cells yielded 12 protein-coding genes (ADM, APBB3, CD160, CFD, CITED2, CTSZ, IER5, NFKBIZ, NR4A2, NUCKS1, SERTAD1, TNF) that were differentially expressed between 29 unmedicated depressed patients with a mood disorder (8 bipolar disorder, 21 major depressive disorder) and 24 healthy controls (HCs). Several of these genes have been implicated in neurological disorders and/or apoptosis. Ingenuity Pathway Analysis yielded two genes networks, one centered around TNF with NFKβ, TGFβ, and ERK as connecting hubs, and the second network indicating cell cycle and/or kinase signaling anomalies. fMRI scanning was conducted using a backward-masking task in which subjects were presented with emotionally-valenced faces. Compared with HCs, the depressed subjects displayed a greater hemodynamic response in the right amygdala, left hippocampus, and the ventromedial prefrontal cortex to masked sad versus happy faces. The mRNA levels of several genes were significantly correlated with the hemodynamic response of the amygdala, vmPFC and hippocampus to masked sad versus happy faces. Differentially-expressed transcripts were significantly correlated with thickness of the left subgenual ACC, and volume of the hippocampus and caudate. Our results raise the possibility that molecular-level immune dysfunction can be mapped onto macro-level neuroimaging abnormalities, potentially elucidating a mechanism by which inflammation leads to depression.
PMCID: PMC3577998  PMID: 23064081
19.  Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2 
The New England journal of medicine  2013;369(25):2391-2405.
Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge.
We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry.
Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients.
Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.)
PMCID: PMC3966280  PMID: 24325359
20.  ALOX5 Polymorphism Associates with Increased Leukotriene Production and Reduced Lung Function and Asthma Control in Children with Poorly Controlled Asthma 
Identification of risk factors for reduced asthma control could improve the understanding and treatment of asthma. A promoter polymorphism in the 5-lipoxygenase gene affects gene expression and response to asthma therapy but its impact on disease control remains unclear.
We sought to determine if the ALOX5 promoter SP1 tandem repeat polymorphism was associated with changes in cysteinyl leukotriene production, lung function, airway inflammation and asthma control score.
We analyzed 270 children 6-17 years old with poorly controlled asthma enrolled in a 6-month clinical trial (NCT00604851). In secondary analysis, we associated the ALOX5 promoter SP1 tandem repeat polymorphism genotype (rs59439148) with asthma outcomes using both additive and recessive genetic models. We evaluated FEV1 percent predicted, symptom control, exhaled nitric oxide and urinary LTE4 levels.
14.8% (40/270) of all children (and 28% (38/135) of African Americans) carried 2 non-5 repeat variant alleles of rs59439148. Children who were homozygous for variant alleles had significantly higher urinary LTE4 levels (38 versus 30 nmol/mol creatinine, p=.0134), significantly worse FEV1% predicted (84 versus 91, p=.017), and a trend toward worse asthma control. FEV1% predicted values were significantly negatively correlated with urinary LTE4 (r = -0.192, p=.009).
Conclusion and Clinical Relevance
Carrying two copies of a minor variant ALOX5 promoter SP1 tandem repeat allele contributes to increased cysLT exposure as determined by urinary LTE4 levels, reduced lung function, and potentially worse asthma control. ALOX5 promoter SP1 tandem repeat genotype may be a risk factor for worse asthma outcomes.
PMCID: PMC3633142  PMID: 23600541
Asthma; Children; ALOX5; 5-Lipoxygenase; Leukotriene; Asthma Control; FEV1; Exhaled Nitric Oxide; Genetic Association
21.  Computed tomography-based diagnosis of diffuse compensatory enlargement of coronary arteries using scaling power laws 
Glagov's positive remodelling in the early stages of coronary atherosclerosis often results in plaque rupture and acute events. Because positive remodelling is generally diffused along the epicardial coronary arterial tree, it is difficult to diagnose non-invasively. Hence, the objective of the study is to assess the use of scaling power law for the diagnosis of positive remodelling of coronary arteries based on computed tomography (CT) images. Epicardial coronary arterial trees were reconstructed from CT scans of six Ossabaw pigs fed on a high-fat, high-cholesterol, atherogenic diet for eight months as well as the same number of body-weight-matched farm pigs fed on a lean chow (101.9±16.1 versus 91.5±13.1 kg). The high-fat diet Ossabaw pig model showed diffuse positive remodelling of epicardial coronary arteries. Good fit of measured coronary data to the length–volume scaling power law ( where Lc and Vc are crown length and volume) were found for both the high-fat and control groups (R2 = 0.95±0.04 and 0.99±0.01, respectively). The coefficient, KLV, decreased significantly in the high-fat diet group when compared with the control (14.6±2.6 versus 40.9±5.6). The flow–length scaling power law, however, was nearly unaffected by the positive remodelling. The length–volume and flow–length scaling power laws were preserved in epicardial coronary arterial trees after positive remodelling. KLV < 18 in the length–volume scaling relation is a good index of positive remodelling of coronary arteries. These findings provide a clinical rationale for simple, accurate and non-invasive diagnosis of positive remodelling of coronary arteries, using conventional CT scans.
PMCID: PMC3627114  PMID: 23365197
coronary compensatory enlargement; scaling power law; computed tomography
22.  RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia 
Nature genetics  2014;46(2):116-125.
The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation.
PMCID: PMC3960636  PMID: 24413735
23.  Advances in fidelity measurement for mental health services research 
Mental health intervention research requires clear and accurate specification of treatment conditions in intervention studies. Measures are increasingly available for community-based interventions for persons with serious mental illnesses. Measures must go beyond structural features to assess critical processes in interventions. They must also balance effectiveness, adequate coverage of active treatment elements, with efficiency, the degree to which measures may be used cost-effectively. The context of their use is changing with the emergence of new frameworks for implementation research and quality improvement.
The focus, content, and results of preliminary studies of four recently developed fidelity measures are described. Measures respectively assess fidelity to case management, cognitive therapy for psychosis, illness management and recovery, and assertive community treatment.
Fidelity measures described assess interventions in a range of treatment contexts from dyads to teams. Each measure focuses assessment resources on the elements critical to the respective intervention. Each has demonstrated coverage of its target intervention and satisfactory psychometric properties and is related to outcomes. Measures have been used for training, quality improvement, or certification. Current fidelity measures assess domains and have uses beyond their nominal position in implementation and quality frameworks.
Process components in community-based interventions can be effectively assessed in fidelity measures. Omission of elements assessing potentially critical, active treatment components poses risk to both research and practice until there is evidence to demonstrate they are non-essential. Further development of fidelity measurement theory and approaches should articulate with development of theory and methods in implementation science.
PMCID: PMC3954528  PMID: 22854723
24.  Signatures of mutational processes in human cancer 
Alexandrov, Ludmil B. | Nik-Zainal, Serena | Wedge, David C. | Aparicio, Samuel A.J.R. | Behjati, Sam | Biankin, Andrew V. | Bignell, Graham R. | Bolli, Niccolo | Borg, Ake | Børresen-Dale, Anne-Lise | Boyault, Sandrine | Burkhardt, Birgit | Butler, Adam P. | Caldas, Carlos | Davies, Helen R. | Desmedt, Christine | Eils, Roland | Eyfjörd, Jórunn Erla | Foekens, John A. | Greaves, Mel | Hosoda, Fumie | Hutter, Barbara | Ilicic, Tomislav | Imbeaud, Sandrine | Imielinsk, Marcin | Jäger, Natalie | Jones, David T.W. | Jones, David | Knappskog, Stian | Kool, Marcel | Lakhani, Sunil R. | López-Otín, Carlos | Martin, Sancha | Munshi, Nikhil C. | Nakamura, Hiromi | Northcott, Paul A. | Pajic, Marina | Papaemmanuil, Elli | Paradiso, Angelo | Pearson, John V. | Puente, Xose S. | Raine, Keiran | Ramakrishna, Manasa | Richardson, Andrea L. | Richter, Julia | Rosenstiel, Philip | Schlesner, Matthias | Schumacher, Ton N. | Span, Paul N. | Teague, Jon W. | Totoki, Yasushi | Tutt, Andrew N.J. | Valdés-Mas, Rafael | van Buuren, Marit M. | van ’t Veer, Laura | Vincent-Salomon, Anne | Waddell, Nicola | Yates, Lucy R. | Zucman-Rossi, Jessica | Futreal, P. Andrew | McDermott, Ultan | Lichter, Peter | Meyerson, Matthew | Grimmond, Sean M. | Siebert, Reiner | Campo, Elías | Shibata, Tatsuhiro | Pfister, Stefan M. | Campbell, Peter J. | Stratton, Michael R.
Nature  2013;500(7463):415-421.
All cancers are caused by somatic mutations. However, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here, we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, kataegis, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer with potential implications for understanding of cancer etiology, prevention and therapy.
PMCID: PMC3776390  PMID: 23945592
25.  Durable adoptive immunotherapy for leukemia produced by manipulation of multiple regulatory pathways of CD8+ T cell tolerance 
Cancer research  2012;73(2):605-616.
Tolerizing mechanisms within the host and tumor microenvironment inhibit T cell effector functions that can control cancer. These mechanisms blunt adoptive immunotherapy with infused T cells due to a complex array of signals that determine T cell tolerance, survival, or deletion. Ligation of the negative regulatory receptors CTLA4, PD-1(PDCD1) or LAG3 on T cells normally hinders their response to antigen through non-redundant biochemical processes that interfere with stimulatory pathways. In this study, we used an established mouse model of T cell tolerance to define the roles of these inhibitory receptors in regulating CD8+ T cell tolerance during adoptive immunotherapy to treat leukemia. Blocking CTLA4 and PD-1 in vivo combined to promote survival of transferred T cells despite powerful deletional signals that mediate Bim (BCL2L11)-dependent apoptosis. However, this dual blockade was not optimal for stimulating effector function by responding T cells, which required the additional blockade of LAG3 to induce full expansion and allow the acquisition of robust cytolytic activity. Thus, the cooperation of multiple distinct regulatory pathways was needed for the survival and effector differentiation of adoptively transferred tumor-reactive CD8+ T cells. Our work defines the immune escape pathways where simultaneous blockade could yield durable immunotherapeutic responses that can eradicate disseminated leukemia.
PMCID: PMC3548961  PMID: 23188506
CD8+; T cells; tolerance; cancer; immunotherapy

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