Publications by authors named "Matthew J Fenton"

50 Publications

Cell-Free DNA in Pediatric Solid Organ Transplantation Using a New Detection Method of Separating Donor-Derived from Recipient Cell-Free DNA.

Clin Chem 2020 Oct;66(10):1300-1309

Department of Paediatric Nephrology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK.

Background: The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients.

Methods: We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available.

Results: The R "genotype-free" method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients.

Conclusion: We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients.
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http://dx.doi.org/10.1093/clinchem/hvaa173DOI Listing
October 2020

The complication rate of intravascular ultrasound (IVUS) in a multicenter pediatric heart transplant population: A study of the international pediatric IVUS consortium.

Clin Transplant 2020 09 28;34(9):e13981. Epub 2020 Jul 28.

Division of Pediatric Cardiology, Loma Linda University Children's Hospital and Medical Center, Loma Linda, California, USA.

Background: Our purpose was to determine the complication rate from intravascular ultrasound (IVUS) in a large, multicenter cohort of pediatric heart transplant (PHT) patients.

Methods: We retrospectively reviewed all PHT who underwent IVUS at 5 institutions (2006-2014). Rates of major and minor complications were calculated. All adverse events (AE) were graded from 1 to 5 using a previously published AE severity scale.

Results: There were 1380 catheterizations in 505 patients and 32 AE (2.3%); 9 major (0.6%) and 23 AE (1.7%). The major AE attributed to IVUS were all coronary artery vasospasm (7). Major and minor AE rates directly related to IVUS were 0.5% and 0.7%, respectively. Minor AE possibly attributable to IVUS included excessive fluoroscopy (3) and transient ST segment changes (7). Of AE related to IVUS, only 3 were of moderate severity. The rest were ≤ minor in severity. There were no reports of coronary artery dissection or death.

Conclusion: Most AE during routine PHT coronary evaluation with IVUS were minor and not directly related to the use of IVUS. The number of coronary related AE was similar to a registry-based report of coronary angiography alone. Efforts to minimize IVUS-related complications should be focused on preventing coronary artery vasospasm.
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http://dx.doi.org/10.1111/ctr.13981DOI Listing
September 2020

The National Institute of Allergy and Infectious Diseases and Scientific Societies Meeting on Research Training Efforts: Summary of Recommendations to Address Early-Stage Investigators.

J Infect Dis 2020 10;222(10):1589-1591

Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

A group of representatives from scientific societies and organizations met to discuss possible solutions for funding and retaining early-stage investigators in research that supports the National Institute of Allergy and Infectious Diseases research agenda. This article describes perspectives voiced during that meeting.
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http://dx.doi.org/10.1093/infdis/jiaa282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552421PMC
October 2020

Outcomes of early NIH-funded investigators: Experience of the National Institute of Allergy and Infectious Diseases.

PLoS One 2018 12;13(9):e0199648. Epub 2018 Sep 12.

Office of the Director, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States of America.

Survival of junior scientists in academic biomedical research is difficult in today's highly competitive funding climate. National Institute of Health (NIH) data on first-time R01 grantees indicate the rate at which early investigators drop out from a NIH-supported research career is most rapid 4 to 5 years from the first R01 award. The factors associated with a high risk of dropping out, and whether these factors impact all junior investigators equally, are unclear. We identified a cohort of 1,496 investigators who received their first R01-equivalent (R01-e) awards from the National Institute of Allergy and Infectious Diseases between 2003 and 2010, and studied all their subsequent NIH grant applications through 2016. Ultimately, 57% of the cohort were successful in obtaining new R01-e funding, despite highly competitive conditions. Among those investigators who failed to compete successfully for new funding (43%), the average time to dropping out was 5 years. Investigators who successfully obtained new grants showed remarkable within-person consistency across multiple grant submission behaviors, including submitting more applications per year, more renewal applications, and more applications to multiple NIH Institutes. Funded investigators appeared to have two advantages over their unfunded peers at the outset: they had better scores on their first R01-e grants and they demonstrated an early ability to write applications that would be scored, not triaged. The cohort rapidly segregated into two very different groups on the basis of PI consistency in the quality and frequency of applications submitted after their first R01-e award. Lastly, we identified a number of specific demographic factors, intitutional characteristics, and grant submission behaviors that were associated with successful outcomes, and assessed their predictive value and relative importance for the likelihood of obtaining additional NIH funding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199648PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135352PMC
February 2019

Potential for and timing of recovery in children with dilated cardiomyopathy.

Int J Cardiol 2018 Sep;266:162-166

Cardiothoracic Unit, Great Ormond Street Hospital for Children NHS Foundation Trust, Great Ormond Street, London WC1N 3JH, UK.

Objective: Understanding the clinical course and time-frame for recovery is helpful to guide management and counselling following a diagnosis of Dilated Cardiomyopathy (DCM). We aimed to document outcomes and time to recovery for a cohort of patients with a dilated cardiomyopathy phenotype.

Methods: An observational cohort methodology was used to collect retrospective data from the departmental database for those identified with DCM. Data relating to mode of presentation, echocardiographic parameters, clinical management and outcome were collated and analysed. Predictors and time-scale for recovery were investigated and reported.

Results: 209 new referrals were included within the time frame. 82 children median age 1.0years (IQR 3.4) required intensive care (ICU) and their survival without death or transplant was 51% to one year and 45% to five years. 127 children presented to the pediatric heart failure clinic. Excluding 58 with neuromuscular disease, median age was 4.1years (IQR 11.3) & survival without death or transplant 85% to 1year and 50% to 5years. NT-proBNP normalized in survivors before echocardiographic parameters. Predictors of recovery included younger age, female sex and smaller left ventricular end diastolic Z score on echocardiogram at presentation.

Conclusion: Transplant-free survival to one year is significantly better for patients presenting to clinic, but longer-term survival is better amongst those presenting to ICU due to a late attrition in those with less severe heart failure at presentation. Falling NT-proBNP is the earliest marker of recovery. Recovery of cardiac function remains possible up to three years from presentation.
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http://dx.doi.org/10.1016/j.ijcard.2017.12.075DOI Listing
September 2018

Heart failure from heart muscle disease in childhood: a 5-10 year follow-up study in the UK and Ireland.

ESC Heart Fail 2016 Jun 24;3(2):107-114. Epub 2016 Jan 24.

Cardiothoracic Unit Great Ormond Street Hospital for Children NHS Foundation Trust London UK.

Aims: Our original study, the first national prospective study of new-onset heart failure from heart muscle disease in children, showed overall 1-year survival of 82%, and event (death or transplantation)-free survival of 66%. This study aimed to evaluate 5 + year outcomes of this important cohort.

Methods And Results: All centres in the UK and Ireland with 1-year event-free survivors participated ( = 14). Anonymised data based on last hospital attendance and echocardiograms were reviewed. The investigator was blinded to outcome at the time of echo review. Of sixty-nine 1-year event-free survivors, data were obtained on 64, with three lost to follow-up and two moved abroad. There were three deaths at 2.2, 3.3 and 9.0 years after presentation and one transplant, at 5.2 years. Overall/event-free survival was 77%/62% at 5 years and 73%/59% at 10 years, respectively. Overall and event-free survival conditional on 1-year survival was 94% at 5 years, and 89% at 10 years. For the 60 event-free survivors, median (range) follow-up duration was 9.04 (5.0-10.33) years for those still under review ( = 45), or time to discharge 5.25 (0.67-10.0) years ( = 15). Fifty-eight were in New York Heart Association (NYHA) Class 1, and two in Class 2. Forty-one out of sixty had normal echocardiograms at last follow-up. Predictors of better longer-term outcome were the same as for the original 1-year follow-up study, namely, younger age and higher fractional shortening measurement at presentation.

Conclusions: Children who survive the first year following their first presentation with significant heart failure from heart muscle disease have a good longer-term outcome although there remains a small attrition rate.
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http://dx.doi.org/10.1002/ehf2.12082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066798PMC
June 2016

Immunobiology of influenza vaccines.

Chest 2013 Feb;143(2):502-510

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

Vaccination is the primary strategy for prevention and control of influenza. The surface hemagglutinin (HA) protein of the influenza virus contains two structural elements (head and stalk) that differ in their potential utility as vaccine targets. The head of the HA protein is the primary target of antibodies that confer protective immunity to influenza viruses. The underlying health status, age, and gene polymorphisms of vaccine recipients and, just as importantly, the extent of the antigenic match between the viruses in the vaccine and those that are circulating modulate influenza vaccine protection. Vaccine adjuvants and live attenuated influenza vaccine improve the breadth of immunity to seasonal and pandemic virus strains. Eliciting antibodies against the conserved HA stem region that cross-react with HAs within influenza virus types or subtypes would allow for the development of a universal influenza vaccine. The highly complex network of interactions generated after influenza infection and vaccination can be studied with the use of systems biology tools, such as DNA microarray chips. The use of systems vaccinology has allowed for the generation of gene expression signatures that represent key transcriptional differences between asymptomatic and symptomatic host responses to influenza infection. Additionally, the use of systems vaccinology tools have resulted in the identification of novel surrogate gene markers that are predictors of the magnitude of host responses to vaccines, which is critical to both vaccine development and public health. Identifying associations between variations in vaccine immune responses and gene polymorphisms is critical in the development of universal influenza vaccines.
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http://dx.doi.org/10.1378/chest.12-1711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619640PMC
February 2013

Summary of the NIAID-sponsored food allergy guidelines.

Am Fam Physician 2012 Jul;86(1):43-50

Olmsted Medical Center, Rochester, Minnesota 55904, USA.

Patients with suspected food allergies are commonly seen in clinical practice. Although up to 15 percent of parents believe their children have food allergies, these allergies have been confirmed in only 1 to 3 percent of all Americans. Family physicians must be able to separate true food allergies from food intolerance, food dislikes, and other conditions that mimic food allergy. The most common foods that produce allergic symptoms are milk, eggs, seafood, peanuts, and tree nuts. Although skin testing and in vitro serum immunoglobulin E assays may help in the evaluation of suspected food allergies, they should not be performed unless the clinical history suggests a specific food allergen to which testing can be targeted. Furthermore, these tests do not confirm food allergy. Confirmation requires a positive food challenge or a clear history of an allergic reaction to a food and resolution of symptoms after eliminating that food from the diet. More than 70 percent of children will outgrow milk and egg allergies by early adolescence, whereas peanut allergies usually remain throughout life. The most serious allergic response to food allergy is anaphylaxis. It requires emergency care that should be initiated by the patient or family using an epinephrine autoinjector, which should be carried by anyone with a diagnosed food allergy. These and other recommendations presented in this article are derived from the Guidelines for the Diagnosis and Management of Food Allergy in the United States, published by the National Institute of Allergy and Infectious Diseases.
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July 2012

Lung dendritic cells at the innate-adaptive immune interface.

J Leukoc Biol 2011 Nov 1;90(5):883-95. Epub 2011 Aug 1.

Department of Pediatrics, National Jewish Health, Denver, CO 80206, USA.

This review updates the basic biology of lung DCs and their functions. Lung DCs have taken center stage as cellular therapeutic targets in new vaccine strategies for the treatment of diverse human disorders, including asthma, allergic lung inflammation, lung cancer, and infectious lung disease. The anatomical distribution of lung DCs, as well as the division of labor between their subsets, aids their ability to recognize and endocytose foreign substances and to process antigens. DCs can induce tolerance in or activate naïve T cells, making lung DCs well-suited to their role as lung sentinels. Lung DCs serve as a functional signaling/sensing unit to maintain lung homeostasis and orchestrate host responses to benign and harmful foreign substances.
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http://dx.doi.org/10.1189/jlb.0311134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206474PMC
November 2011

Innate immunity in allergic disease.

Immunol Rev 2011 Jul;242(1):106-27

Asthma, Allergy and Inflammation Branch, Division of Allergy, Immunology, and Transplantation, Department of Health and Human Services, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-6601, USA.

The innate immune system consists of multiple cell types that express germline-encoded pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Allergens are frequently found in forms and mixtures that contain PAMPs and DAMPs. The innate immune system is interposed between the external environment and the internal acquired immune system. It is also an integral part of the airways, gut, and skin. These tissues face continuous exposure to allergens, PAMPs, and DAMPs. Interaction of allergens with the innate immune system normally results in immune tolerance but, in the case of allergic disease, this interaction induces recurring and/or chronic inflammation as well as the loss of immunologic tolerance. Upon activation by allergens, the innate immune response commits the acquired immune response to a variety of outcomes mediated by distinct T-cell subsets, such as T-helper 2, regulatory T, or T-helper 17 cells. New studies highlighted in this review underscore the close relationship between allergens, the innate immune system, and the acquired immune system that promotes homeostasis versus allergic disease.
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http://dx.doi.org/10.1111/j.1600-065X.2011.01025.xDOI Listing
July 2011

Guidelines for the Diagnosis and Management of Food Allergy in the United States: summary of the NIAID-sponsored expert panel report.

J Am Diet Assoc 2011 Jan;111(1):17-27

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Department of Medicine Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1016/j.jada.2010.10.033DOI Listing
January 2011

Guidelines for the diagnosis and management of food allergy in the United States: report of the NIAID-sponsored expert panel.

J Allergy Clin Immunol 2010 Dec;126(6 Suppl):S1-58

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA, USA.

Food allergy is an important public health problem that affects children and adults and may be increasing in prevalence. Despite the risk of severe allergic reactions and even death, there is no current treatment for food allergy: the disease can only be managed by allergen avoidance or treatment of symptoms. The diagnosis and management of food allergy also may vary from one clinical practice setting to another. Finally, because patients frequently confuse nonallergic food reactions, such as food intolerance, with food allergies, there is an unfounded belief among the public that food allergy prevalence is higher than it truly is. In response to these concerns, the National Institute of Allergy and Infectious Diseases, working with 34 professional organizations, federal agencies, and patient advocacy groups, led the development of clinical guidelines for the diagnosis and management of food allergy. These Guidelines are intended for use by a wide variety of health care professionals, including family practice physicians, clinical specialists, and nurse practitioners. The Guidelines include a consensus definition for food allergy, discuss comorbid conditions often associated with food allergy, and focus on both IgE-mediated and non-IgE-mediated reactions to food. Topics addressed include the epidemiology, natural history, diagnosis, and management of food allergy, as well as the management of severe symptoms and anaphylaxis. These Guidelines provide 43 concise clinical recommendations and additional guidance on points of current controversy in patient management. They also identify gaps in the current scientific knowledge to be addressed through future research.
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http://dx.doi.org/10.1016/j.jaci.2010.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241964PMC
December 2010

Vaccination of patients with mild and severe asthma with a 2009 pandemic H1N1 influenza virus vaccine.

J Allergy Clin Immunol 2011 Jan 9;127(1):130-7, 137.e1-3. Epub 2010 Dec 9.

University of Wisconsin School of Medicine and Public Health, Madison, Wis., USA.

Background: Asthma was the most common comorbidity of patients hospitalized with 2009 H1N1 influenza.

Objective: We sought to assess the immunogenicity and safety of an unadjuvanted, inactivated 2009 H1N1 vaccine in patients with severe versus mild-to-moderate asthma.

Methods: We conducted an open-label study involving 390 participants (age, 12-79 years) enrolled in October-November 2009. Severe asthma was defined as need for 880 μg/d or more of inhaled fluticasone equivalent, systemic corticosteroids, or both. Within each severity group, participants were randomized to receive intramuscularly 15 or 30 μg of 2009 H1N1 vaccine twice 21 days apart. Immunogenicity end points were seroprotection (hemagglutination inhibition assay titer ≥40) and seroconversion (4-fold or greater titer increase). Safety was assessed through local and systemic reactogenicity, asthma exacerbations, and pulmonary function.

Results: In patients with mild-to-moderate asthma (n = 217), the 2009 H1N1 vaccine provided equal seroprotection 21 days after the first immunization at the 15-μg (90.6%; 95% CI, 83.5% to 95.4%) and 30-μg (95.3%; 95% CI, 89.4% to 98.5%) doses. In patients with severe asthma (n = 173), seroprotection 21 days after the first immunization was 77.9% (95% CI, 67.7% to 86.1%) and 94.1% (95% CI, 86.8% to 98.1%) at the 15- and 30-μg doses, respectively (P = .004). The second vaccination did not provide further increases in seroprotection. Participants with severe asthma who are older than 60 years showed the lowest seroprotection (44.4% at day 21) with the 15-μg dose but had adequate seroprotection with 30 μg. The 2 dose groups did not differ in seroconversion rates. There were no safety concerns.

Conclusion: Monovalent inactivated 2009 H1N1 pandemic influenza vaccine was safe and provided overall seroprotection as a surrogate of efficacy. In patients older than 60 years with severe asthma, a 30-μg dose might be more appropriate.
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http://dx.doi.org/10.1016/j.jaci.2010.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017653PMC
January 2011

Asthma in the inner city: the perspective of the National Institute of Allergy and Infectious Diseases.

J Allergy Clin Immunol 2010 Mar;125(3):540-4

Asthma and Inflammation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md 20892, USA.

Since 1991, the National Institute of Allergy and Infectious Diseases (NIAID) has funded four consecutive research initiatives to investigate the problem of high asthma prevalence, morbidity and mortality in poor urban communities. The multi-site studies conducted under these initiatives have identified key risk factors for asthma morbidity and novel interventions to improve asthma control. NIAID focuses its asthma and allergy programs on understanding the interaction of the immune system with allergens and infectious agents and identifying genetic and epigenetic elements that influence the immune system. A key goal in this field is to define mechanisms of immune system deviation and immune tolerance and apply this knowledge to generate improvements in asthma care and allergen immunotherapy. A related goal is to further understand the environmental, social, and immunological elements that impact on the development of inner-city asthma through in-depth characterization and longitudinal follow-up of inner-city children from the time of birth. In the past 5 years, NIH budgetary constraints have imposed many challenges for the academic research community. Despite these constraints, NIAID has maintained its support of a highly productive asthma and allergy research program.
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http://dx.doi.org/10.1016/j.jaci.2010.01.040DOI Listing
March 2010

TLR2 engagement on dendritic cells promotes high frequency effector and memory CD4 T cell responses.

J Immunol 2009 Dec;183(12):7832-41

Department of Surgery and Microbiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Ligation of TLR by distinct pathogen components provides essential signals for T cell priming, although how individual TLR engagement affects primary and memory T cell responses is not well defined. In this study, we demonstrate distinct effects of TLR2 vs TLR4 engagement on primary and memory CD4 T cell responses due to differential effects on APC. Priming of influenza hemagglutinin (HA)-specific naive CD4 T cells with HA peptide and the TLR2 agonist Pam3CysK in vivo resulted in a high frequency of activated HA-specific CD4 T cells that predominantly produced IL-2 and IL-17, whereas priming with HA peptide and the TLR4 agonist LPS yielded a lower frequency of HA-specific CD4 T cells and predominant IFN-gamma producers. TLR2 agonist priming depended on TLR2 expression by APC, as wild-type CD4 T cells did not expand in response to peptide and Pam3CysK in TLR2-deficient hosts. TLR2-mediated priming also led to an increased frequency of Ag-specific memory CD4 T cells compared with TLR4 priming and mediated enhanced secondary responses to influenza challenge. Our results show that TLR engagement on APC influences both primary and secondary CD4 T cell responses, and suggest that long-term functional capacities of T cells are set by innate signals during early phases of an infection.
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http://dx.doi.org/10.4049/jimmunol.0901683DOI Listing
December 2009

Summary of the 2008 National Institute of Allergy and Infectious Diseases-US Food and Drug Administration Workshop on Food Allergy Clinical Trial Design.

J Allergy Clin Immunol 2009 Oct 27;124(4):671-8.e1. Epub 2009 Jun 27.

Division of Allergy, Immunology and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20892-6601, USA.

This article summarizes the proceedings of a 2008 Workshop on Food Allergy Clinical Trials Design co-organized by the National Institute of Allergy and Infectious Diseases and the US Food and Drug Administration. The use of food allergens both as therapy and for oral food challenges is associated with a risk of anaphylaxis. Investigators are strongly encouraged to address regulatory considerations by discussing proposed studies with the US Food and Drug Administration. Food allergen administration through the oral or sublingual routes might be less risky than through the subcutaneous route, but this hypothesis has not been proved, and subjects with food allergy might still be at high risk of allergic reactions to such allergen administration. Two distinct mechanisms might lead to beneficial clinical outcomes: desensitization (reversible when food allergen therapy is stopped) and tolerance (persistent benefit even after allergen therapy is stopped). There are important clinical distinctions between desensitization and tolerance. The efficacy of a therapy for food allergy can be evaluated by assessing changes in the dose response to double-blind, placebo-controlled oral food challenges before and after therapy and also by assessing changes in the number of allergic episodes during a longitudinal natural history/exposure study; both approaches have strengths and limitations.
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http://dx.doi.org/10.1016/j.jaci.2009.05.027DOI Listing
October 2009

Involvement of TLR2 and TLR4 in inflammatory immune responses induced by fine and coarse ambient air particulate matter.

J Leukoc Biol 2009 Aug;86(2):303-12

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

Induction of proinflammatory mediators by alveolar macrophages exposed to ambient air particulate matter has been suggested to be a key factor in the pathogenesis of inflammatory and allergic diseases in the lungs. However, receptors and mechanisms underlying these responses have not been fully elucidated. In this study, we examined whether TLR2, TLR4, and the key adaptor protein, MyD88, mediate the expression of proinflammatory cytokines and chemokines by mouse peritoneal macrophages exposed to fine and coarse PM. TLR2 deficiency blunted macrophage TNF-alpha and IL-6 expression in response to fine (PM2.5), while not affecting cytokine-inducing ability of coarse NIST Standard Reference Material (SRM 1648) particles. In contrast, TLR4(-/-) macrophages showed inhibited cytokine expression upon stimulation with NIST SRM 1648 but exhibited normal responses to PM2.5. Preincubation with polymyxin B markedly suppressed the capacity of NIST SRM 1648 to elicit TNF-alpha and IL-6, indicating endotoxin as a principal inducer of cytokine responses. Overexpression of TLR2 in TLR2/4-deficient human embryonic kidney 293 cells imparted PM2.5 sensitivity, as judged by IL-8 gene expression, whereas NIST SRM 1648, but not PM2.5 elicited IL-8 expression in 293/TLR4/MD-2 transfectants. Engagement of TLR4 by NIST SRM 1648 induced MyD88-independent expression of the chemokine RANTES, while TLR2-reactive NIST IRM PM2.5 failed to up-regulate this response. Consistent with the shared use of MyD88 by TLR2 and TLR4, cytokine responses of MyD88(-/-) macrophages to both types of air PM were significantly reduced. These data indicate differential utilization of TLR2 and TLR4 but shared use of MyD88 by fine and coarse air pollution particles.
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http://dx.doi.org/10.1189/jlb.1008587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726765PMC
August 2009

TLR4/MyD88/PI3K interactions regulate TLR4 signaling.

J Leukoc Biol 2009 Jun 16;85(6):966-77. Epub 2009 Mar 16.

University of Maryland, Baltimore, 660 W. Redwood Street, Room 324, Baltimore, MD 21201, USA.

TLRs activate immune responses by sensing microbial structures such as bacterial LPS, viral RNA, and endogenous "danger" molecules released by damaged host cells. MyD88 is an adapter protein that mediates signal transduction for most TLRs and leads to activation of NF-kappaB and MAPKs and production of proinflammatory cytokines. TLR4-mediated signaling also leads to rapid activation of PI3K, one of a family of kinases involved in regulation of cell growth, apoptosis, and motility. LPS stimulates phosphorylation of Akt, a downstream target of PI3K, in wild-type (WT) mouse macrophages. LPS-induced phosphorylation of Akt serine 473 was blunted in MyD88(-/-) macrophages and was completely TLR4-dependent. MyD88 and p85 were shown previously to co-immunoprecipitate, and a YXXM motif within the Toll-IL-1 resistance (TIR) domain of MyD88 was suggested to be important for this interaction. To test this hypothesis, we compared expressed MyD88 variants with mutations within the YXXM motif or lacking the TIR domain or death domain and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co-immunoprecipitation analyses. The YXXM --> YXXA mutant MyD88 bound more strongly to p85, TLR4, and WT MyD88 than the other variants, yet was significantly less active than WT MyD88, suggesting that sustained interaction of MyD88/PI3K with the TLR4 intracellular "signaling platform" negatively regulates signaling. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of the PI3K substrate, thereby negatively regulating signaling.
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http://dx.doi.org/10.1189/jlb.1208763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698589PMC
June 2009

New-onset heart failure due to heart muscle disease in childhood: a prospective study in the United kingdom and Ireland.

Circulation 2008 Jan 17;117(1):79-84. Epub 2007 Dec 17.

Department of Congenital Heart Disease, Evelina Children's Hospital, Guys' and St Thomas' NHS Trust, London, United Kingdom.

Background: We undertook the first prospective, national, multicenter study to describe the incidence and outcome of heart muscle disease-induced heart failure in children.

Methods And Results: Data were collected on patients admitted to a hospital through 2003 with a first episode of heart failure in the absence of congenital heart disease. All 17 pediatric cardiac centers in the United Kingdom and Ireland participated. Follow-up data were obtained to a minimum of 1 year. The incidence was 0.87/100,000 population <16 years (n=104; 53 girls; 95% confidence interval 0.71 to 1.05 per 100,000). Median age at presentation was 1 year, with 82% in New York Heart Association class III to IV. Causes of heart failure included dilated cardiomyopathy (50 idiopathic, 8 familial), probable myocarditis (23), occult arrhythmia (7), anthracycline toxicity (5), metabolic disease (4), left ventricular noncompaction (3), and other (4). Overall 1-year survival was 82%, and event (death or transplantation)-free survival was 66%. Regression analysis showed older age and reduced systolic function on admission echocardiogram increased the event risk. Only 8% of event-free survivors (n=69) remained in New York Heart Association class III to IV, but 35 required readmission during the study period, and all but 8 remained on medication.

Conclusions: This first national prospective study of new-onset heart failure in children has shown an incidence of 0.87/100,000. Multivariable analysis of survival data indicates a better outcome for younger children and for those with better systolic function at presentation, but overall, one third of children die or require transplantation within 1 year of presentation.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.106.671735DOI Listing
January 2008

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance.

J Biol Chem 2007 Jun 28;282(22):16042-53. Epub 2007 Mar 28.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.
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http://dx.doi.org/10.1074/jbc.M606781200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675888PMC
June 2007

Positive pretransplantation cytomegalovirus serology is a risk factor for cardiac allograft vasculopathy in children.

Circulation 2007 Apr 12;115(13):1798-805. Epub 2007 Mar 12.

Paediatric Cardiology, Great Ormond Street Hospital, London, United Kingdom.

Background: Cytomegalovirus (CMV) infection has been implicated as a cause of posttransplantation coronary artery disease in adults. The purpose of this retrospective observational study was to evaluate the effect of CMV on outcome after heart transplantation in children.

Methods And Results: Risk factors tested were recipient age, sex, and pretransplantation CMV serology; use of anti-CMV prophylaxis; posttransplantation evidence of CMV infection; and donor CMV serology. Transplantations were stratified traditionally according to CMV risk as low risk (recipient negative/donor negative), intermediate risk (recipient positive), and high risk (recipient negative/donor positive). Primary outcome measures were (1) development of coronary artery vasculopathy, (2) mortality (or graft loss) that occurred outside the early postoperative period, and (3) death (or graft loss) due to vasculopathy. Analysis was by proportional hazards modeling. A total of 165 children underwent heart transplantation, with a mean age at transplantation of 7.8 (SD 5.6) years. Thirty-two children had laboratory evidence of CMV infection after transplantation, but only 6 developed CMV disease or syndrome. Traditional CMV risk stratification correlated well with CMV infection but did not predict mortality, coronary artery disease, or coronary death. In contrast, positive recipient CMV was the only independent predictor of all 3 outcome measures: coronary artery disease (hazard ratio=3.6), all-cause mortality (partial hazard ratio=4.1), and coronary death (hazard ratio=4.6).

Conclusions: In children, pretransplantation recipient CMV status is a more powerful predictor for the development of clinically significant vasculopathy and subsequent death than traditional risk stratification. This phenomenon warrants further investigation.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.106.627570DOI Listing
April 2007

Role of Bacillus anthracis spore structures in macrophage cytokine responses.

Infect Immun 2007 May 5;75(5):2351-8. Epub 2007 Mar 5.

Center for Vaccine Development, Department of Medicine, University of Maryland, 685 W. Baltimore Street, HSF I-480, Baltimore, MD 21201, USA.

The innate immune response of macrophages (Mphi) to spores, the environmentally acquired form of Bacillus anthracis, is poorly characterized. We therefore examined the early Mphi cytokine response to B. anthracis spores, before germination. Mphi were exposed to bacilli and spores of Sterne strain 34F2 and its congenic nongerminating mutant (DeltagerH), and cytokine expression was measured by real-time PCR and an enzyme-linked immunosorbent assay. The exosporium spore layer was retained (exo+) or removed by sonication (exo-). Spores consistently induced a strong cytokine response, with the exo- spores eliciting a two- to threefold-higher response than exo+ spores. The threshold for interleukin-1beta (IL-1beta) production by wild-type Mphi was significantly lower than that required for tumor necrosis factor alpha expression. Cytokine production was largely dependent on MyD88, suggesting Toll-like receptor involvement; however, the expression of beta interferon in MyD88-/- Mphi suggests involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis spore is not immunologically inert, (ii) the exosporium masks epitopes recognized by the Mphi, (iii) the Mphi cytokine response to B. anthracis involves multiple pattern recognition receptors and signaling pathways, and (iv) compared to other cytokines, IL-1beta is expressed at a lower spore concentration.
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http://dx.doi.org/10.1128/IAI.01982-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865778PMC
May 2007

Cutting Edge: Differential inhibition of TLR signaling pathways by cell-permeable peptides representing BB loops of TLRs.

J Immunol 2007 Mar;178(5):2655-60

Department of Microbiology and Immunology, University of Maryland-Baltimore, 660 West Redwood Street, Baltimore, MD 21201, USA.

We designed cell-penetrating peptides comprised of the translocating segment of Drosophila antennapedia homeodomain fused with BB loop sequences of TLR2, TLR4, and TLR1/6. TLR2- and TLR4-BB peptides (BBPs) inhibited NF-kappaB translocation and early IL-1beta mRNA expression induced by LPS, and the lipopeptides S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH (P3C) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-Cys-Ser-Lys(4)-OH (P2C). TLR4- and TLR2-BBPs also strongly inhibited LPS-induced activation of ERK. Only TLR2-BBP significantly inhibited ERK activation induced by P3C, which acts via TLR2/1 heterodimers. BBPs did not inhibit activation of ERK induced by P2C, a TLR2/6 agonist. The TLR2-BBP induced weak activation of p38, but not ERK or cytokine mRNA. The TLR1/6-BBP failed to inhibit NF-kappaB or MAPK activation induced by any agonist. Our results suggest that the receptor BBPs selectively affect different TLR signaling pathways, and that the BB loops of TLR1/6 and TLR2 play distinct roles in formation of receptor heterodimers and recruitment of adaptor proteins.
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http://dx.doi.org/10.4049/jimmunol.178.5.2655DOI Listing
March 2007

TLR2- and TLR4-dependent activation of STAT1 serine phosphorylation in murine macrophages is protein kinase C-delta-independent.

J Endotoxin Res 2006 ;12(4):231-40

Division of Pulmonary and Critical Care Medicine and Mucosal Biology Research Center, University of Maryland School of Medicine, Baltimore, USA.

Engagement of Toll-like receptor (TLR) proteins activates multiple signal transduction pathways. Previous studies demonstrated that TLR2 and TLR4 engagement leads to rapid phosphorylation of the transcription factor STAT1 at serine 727 (Ser-727 STAT1) in murine macrophages. Only TLR4 engagement induced STAT1 phosphorylation at tyrosine 701, although this response was delayed compared with Ser-727 STAT1 phosphorylation. Unlike other cell types, the p38 mitogen-activated protein kinase was necessary, but not sufficient, for TLR-induced phosphorylation of Ser-727 STAT1 in macrophages. We and others had previously shown that Ser-727 STAT1 phosphorylation could be blocked by rottlerin, an inhibitor of protein kinase C-delta (PKC-delta). Here we report that peritoneal exudate macrophages from PKC-delta-deficient mice can be activated through TLR2 and TLR4 to elicit rapid phosphorylation of Ser-727 STAT1, which was blocked by both rottlerin and the p38 inhibitor SB203580, but not by the pan-PKC inhibitor bisindoylmaleamide. Furthermore, both normal and PKC-delta-deficient macrophages secreted comparable amounts of IL-6, IP-10, and RANTES following TLR engagement. In contrast, IFN-gamma-induced STAT1 serine phosphorylation was independent of both PKC-delta and p38. Overall, these studies demonstrate that a PKC-delta-independent signaling pathway downstream of both TLR2 and TLR4 is necessary for Ser-727 STAT1 phosphorylation in primary murine macrophages.
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http://dx.doi.org/10.1179/096805106X102219DOI Listing
September 2006

HMGB1 signals through toll-like receptor (TLR) 4 and TLR2.

Shock 2006 Aug;26(2):174-9

Laboratories of Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.

In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.
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http://dx.doi.org/10.1097/01.shk.0000225404.51320.82DOI Listing
August 2006

Hyaluronan fragments act as an endogenous danger signal by engaging TLR2.

J Immunol 2006 Jul;177(2):1272-81

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 1830 East Monument Street, Baltimore, MD 21205, USA.

Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Czeta-, and NF-kappaB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2(null) mice.
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http://dx.doi.org/10.4049/jimmunol.177.2.1272DOI Listing
July 2006