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

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.

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

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.

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

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.

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

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.

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

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.

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

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 (n = 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 (n = 45), or time to discharge 5.25 (0.67-10.0) years (n = 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.

Immunobiology of influenza vaccines.

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.

Summary of the NIAID-sponsored food allergy guidelines.

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.

Lung dendritic cells at the innate-adaptive immune interface.

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.

Innate immunity in allergic disease.

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.

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

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.

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

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.

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

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.

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

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.

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

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.